In silico, in vitro and in vivo safety evaluation of Limosilactobacillus reuteri strains ATCC PTA-126787 & ATCC PTA-126788 for potential probiotic applications.

The last two decades have witnessed a tremendous growth in probiotics and in the numbers of publications on their potential health benefits. Owing to their distinguishing beneficial effects and long history of safe use, species belonging to the Lactobacillus genus are among the most widely used probiotic species in human food and dietary supplements and are finding increased use in animal feed. Here, we isolated, identified, and evaluated the safety of two novel Limosilactobacillus reuteri (L. reuteri) isolates, ATCC PTA-126787 & ATCC PTA-126788. More specifically, we sequenced the genomes of these two L. reuteri strains using the PacBio sequencing platform. Using a combination of biochemical and genetic methods, we identified the two strains as belonging to L. reuteri species. Detailed in silico analyses showed that the two strains do not encode for any known genetic sequences of concern for human or animal health. In vitro assays confirmed that the strains are susceptible to clinically relevant antibiotics and do not produce potentially harmful by-products such as biogenic amines. In vitro bile and acid tolerance studies demonstrated that the two strains have similar survival profiles as the commercial L. reuteri probiotic strain DSM 17938. Most importantly, daily administration of the two probiotic strains to broiler chickens in drinking water for 26 days did not induce any adverse effect, clinical disease, or histopathological lesions, supporting the safety of the strains in an in vivo avian model. All together, these data provide in silico, in vitro and in vivo evidence of the safety of the two novel candidates for potential probiotic applications in humans as well as animals.

In vivo assessment and characterization of lactic acid bacteria with probiotic profile isolated from human milk powder / Evaluación y caracterización in vivo de bacterias acidolácticas con perfil probiótico aisladas a partir de leche materna en polvo

INTRODUCTION: breast milk (MH) contains nutrients and bioactive compounds for child development, including probiotic bacteria, which contribute to intestinal maturation. This benefit accompanies the individual until adulthood. There are new methods such as spray drying that give this compound a good conservation without loss of microbiota. OBJECTIVE: the aim of this study was to analyze the viability of lactic acid bacteria isolated from human milk with probiotic potential after the spray drying process, as well as to evaluate the possible adhesion in the colon of mice of the Balb/C strain after feeding them powdered human milk and a commercial formula milk. METHOD: we isolated and identified the presence of lactic acid bacteria with possible probiotic potential in powdered human milk using the MALDI-TOF MS technique. Powdered human milk and a commercial formula milk were fed to mice of the Bald/C strain for 14 weeks. Glucose level and weight were measured in the mice. The feces were collected to verify the presence of lactic bacteria. The mice were sacrificed and their intestines were weighed, isolating the lactic acid bacteria both from the intestines and from the feces. The strains isolated from mice fed human milk were evaluated for their probiotic potential, analyzing their ability to inhibit pathogens, resistance to pH, temperature, adhesion, and hydrophobicity. RESULTS: the presence of Lactobacillus fermentum LH01, Lactobacillus rhamnosus LH02, Lactobacullis reuteri LH03, and Lactobacillus plantarum LH05 in powdered human milk was identified. All strains showed a possible probiotic profile due to the ability of bacteria to resist low pH, bile salts, and exposure to gastric enzymes, as well as their hydrophobicity and self-aggregation capacity, and their failure to show hemagglutination or hemolysis activity in a culture medium rich in erythrocytes. We observed that the consumption of powdered human milk prevented weight gain and constipation in mice. CONCLUSIONS: after spray drying, strains with possible probiotic potential may be preserved in human milk. The consumption of powdered human milk with probiotic bacteria prevents constipation and weight gain in mice, when compared to those fed a commercial formula milk

INTRODUCCIÓN: la leche materna (HM) contiene los nutrientes y compuestos bioactivos necesarios para el desarrollo infantil, incluidas bacterias probióticas, que contribuyen a la maduración intestinal. OBJETIVO: el objetivo de este estudio fue analizar la viabilidad de las bacterias acidolácticas aisladas de la leche humana con potencial probiótico, después del proceso de secado, así como evaluar su posible adhesión en el colón de ratones (BAlb/C) alimentados con leche humana en polvo y leche de una fórmula comercial. MÉTODO: se aislaron e identificaron mediante la técnica de Maldi-Tof-MS las bacterias acidolácticas con posible potencial probiótico en la leche humana en polvo. Se alimentó con leche humana en polvo y leche de una fórmula comercial a ratones de la cepa Bald/C durante 14 semanas. Se midieron el nivel de glucosa y el peso. Las heces se recolectaron para verificar la presencia de bacterias lácticas. Los ratones se sacrificaron y se pesaron los intestinos, aislando las bacterias lácticas tanto de los intestinos como de las heces. En las cepas aisladas de la leche humana se evaluó el potencial probiótico analizando su capacidad para inhibir patógenos, resistir distintos pH y temperaturas, adherirse y mostrar hidrofobicidad. RESULTADOS: se identificó la presencia de Lactobacillus fermentum LH01, Lactobacillus rhamnosus LH02, Lactobacullis reuteri LH03 y L. plantarum LH05 en la leche humana en polvo. Todas las cepas mostraron resistencia a los pH bajos, a las sales biliares y a la exposición a enzimas gástricas, así como una buena hidrofobicidad y capacidad de autoagregación. Además, no presentaron actividad de hemaglutinación o hemólisis en un medio de cultivo rico en eritrocitos. Observamos que el consumo de leche humana en polvo evita en los ratones el aumento de peso y el estreñimiento. CONCLUSIONES: después del secado por aspersión, las cepas con posible potencial probiótico pueden conservarse en la leche materna. El consumo de leche humana en polvo con bacterias probióticas evita el estreñimiento y el aumento de peso en los ratones, en comparación con los alimentados con leche de una formula comercial

Characterization of Lactobacillus reuteri WQ-Y1 with the ciprofloxacin degradation ability.

OBJECT: As a broad-spectrum fluoroquinolone antibiotic drug, ciprofloxacin (CIP) is frequently used in the treatment of a wide variety of infections. However, the residues of this antibiotic pose a big threat to the aquatic environment and human health. In this research, Lactobacillus reuteri WQ-Y1 with CIP degradation ability was screened and identified. RESULTS: L. reuteri WQ-Y1 with a degradation rate of 65.1% for 4 µg mL-1 CIP was screened from 17 lactic acid bacteria (LAB), and cytochrome P450 enzyme was confirmed to promote the degradation of CIP by L. reuteri WQ-Y1. Meanwhile, the CIP degradation rate were also higher in 48 h' culture time when co-cultured with 1 mg/mL of glucose in the culture media. Furthermore, result also proved that fluoroquinolone antibiotics with the similar piperazine ring structures could be degraded by L. reuteri WQ-Y1. CONCLUSIONS: L. reuteri WQ-Y1 could degrade fluoroquinolone antibiotics with the similar piperazine ring structure. However, future work still needs to be done on the confirmation of the key enzymes in the cytochrome P450 enzymes family in the biodegradation. The isolated ciprofloxacin-degrading strain L. reuteri WQ-Y1 had a CIP degradation rate of 65.1% at 24 hours, and one biodegradation metabolite was identified and proved to be an important metabolite of CIP from cytochrome P450 enzymes family hydrolysis with UPLC-MS/MS spectrograms approach.

Lactobacillus reuteri AN417 cell-free culture supernatant as a novel antibacterial agent targeting oral pathogenic bacteria.

Lactobacillus reuteri AN417 is a newly characterized probiotic strain. The activity of AN417 against oral pathogenic bacteria is unknown. We investigated the antibacterial activity of cell-free L. reuteri AN417 culture supernatant (LRS) against three oral pathogens: Porphyromonas gingivalis, Fusobacterium nucleatum, and Streptococcus mutans. P. gingivalis and F. nucleatum have been implicated in periodontal disease, whereas S. mutans causes dental caries. Exposing these oral pathogenic bacteria to LRS significantly reduced their growth rates, intracellular ATP levels, cell viability, and time-to-kill. The minimal inhibitory volume of LRS was 10% (v/v) against P. gingivalis, 20% (v/v) for F. nucleatum, and 30% (v/v) for S. mutans. LRS significantly reduced the integrity of biofilms and significantly suppressed the expression of various genes involved in P. gingivalis biofilm formation. The L. reuteri AN417 genome lacked genes encoding reuterin, reuteran, and reutericyclin, which are major antibacterial compounds produced in L. reuteri strains. LRS treated with lipase and α-amylase displayed decreased antibacterial activity against oral pathogens. These data suggest that the antibacterial substances in LRS are carbohydrates and/or fatty acid metabolites. Our results demonstrate that LRS has antimicrobial activity against dental pathogenic bacteria, highlighting its potential utility for the prevention and treatment of P. gingivalis periodontal disease.

Real-Time PCR Assays for the Specific Identification of Probiotic Strains Lactobacillus gasseri BNR17 and Lactobacillus reuteri LRC (NCIMB 30242).

The broad spectrum of health benefits attributed to probiotics has contributed to a rapid increase in the value of the probiotic market. Probiotic health benefits can be strain specific. Thus, strain-level identification of probiotic strains is of paramount importance to ensure probiotic efficacy. Both Lactobacillus gasseri BNR17 and Lactobacillus reuteri LRC (NCIMB 30242) strains have clinically proven health benefits; however, no assays were developed to enable strain-level identification of either of these strains. The objective of this study is to develop strain-specific PCR-based methods for Lactobacillus gasseri BNR17 and Lactobacillus reuteri LRC strains, and to validate these assays according to the guidelines for validating qualitative real-time PCR assays. Using RAST (Rapid Annotation using Subsystem Technology), unique sequence regions were identified in the genome sequences of both strains. Probe-based assays were designed and validated for specificity, sensitivity, efficiency, repeatability, and reproducibility. Both assays were specific to target strain with 100% true positive and 0% false positive rates. Reaction efficiency for both assays was in the range of 90 to 108% with R square values > 0.99. Repeatability and reproducibility were evaluated using five samples at three DNA concentrations each and relative standard deviation was < 4% for repeatability and < 8% for reproducibility. Both of the assays developed and validated in this study for the specific identification of Lactobacillus gasseri BNR17 and Lactobacillus reuteri LRC strains are specific, sensitive, and precise. These assays can be applied to evaluate and ensure compliance in probiotic products.

In vivo assessment and characterization of lactic acid bacteria with probiotic profile isolated from human milk powder.

INTRODUCTION: Introduction: breast milk (MH) contains nutrients and bioactive compounds for child development, including probiotic bacteria, which contribute to intestinal maturation. This benefit accompanies the individual until adulthood. There are new methods such as spray drying that give this compound a good conservation without loss of microbiota. Objective: the aim of this study was to analyze the viability of lactic acid bacteria isolated from human milk with probiotic potential after the spray drying process, as well as to evaluate the possible adhesion in the colon of mice of the Balb/C strain after feeding them powdered human milk and a commercial formula milk. Method: we isolated and identified the presence of lactic acid bacteria with possible probiotic potential in powdered human milk using the MALDI-TOF MS technique. Powdered human milk and a commercial formula milk were fed to mice of the Bald/C strain for 14 weeks. Glucose level and weight were measured in the mice. The feces were collected to verify the presence of lactic bacteria. The mice were sacrificed and their intestines were weighed, isolating the lactic acid bacteria both from the intestines and from the feces. The strains isolated from mice fed human milk were evaluated for their probiotic potential, analyzing their ability to inhibit pathogens, resistance to pH, temperature, adhesion, and hydrophobicity. Results: the presence of Lactobacillus fermentum LH01, Lactobacillus rhamnosus LH02, Lactobacullis reuteri LH03, and Lactobacillus plantarum LH05 in powdered human milk was identified. All strains showed a possible probiotic profile due to the ability of bacteria to resist low pH, bile salts, and exposure to gastric enzymes, as well as their hydrophobicity and self-aggregation capacity, and their failure to show hemagglutination or hemolysis activity in a culture medium rich in erythrocytes. We observed that the consumption of powdered human milk prevented weight gain and constipation in mice. Conclusions: after spray drying, strains with possible probiotic potential may be preserved in human milk. The consumption of powdered human milk with probiotic bacteria prevents constipation and weight gain in mice, when compared to those fed a commercial formula milk.

INTRODUCCIÓN: Introducción: la leche materna (HM) contiene los nutrientes y compuestos bioactivos necesarios para el desarrollo infantil, incluidas bacterias probióticas, que contribuyen a la maduración intestinal. Objetivo: el objetivo de este estudio fue analizar la viabilidad de las bacterias acidolácticas aisladas de la leche humana con potencial probiótico, después del proceso de secado, así como evaluar su posible adhesión en el colón de ratones (BAlb/C) alimentados con leche humana en polvo y leche de una fórmula comercial. Método: se aislaron e identificaron mediante la técnica de Maldi-Tof-MS las bacterias acidolácticas con posible potencial probiótico en la leche humana en polvo. Se alimentó con leche humana en polvo y leche de una fórmula comercial a ratones de la cepa Bald/C durante 14 semanas. Se midieron el nivel de glucosa y el peso. Las heces se recolectaron para verificar la presencia de bacterias lácticas. Los ratones se sacrificaron y se pesaron los intestinos, aislando las bacterias lácticas tanto de los intestinos como de las heces. En las cepas aisladas de la leche humana se evaluó el potencial probiótico analizando su capacidad para inhibir patógenos, resistir distintos pH y temperaturas, adherirse y mostrar hidrofobicidad. Resultados: se identificó la presencia de Lactobacillus fermentum LH01, Lactobacillus rhamnosus LH02, Lactobacullis reuteri LH03 y L. plantarum LH05 en la leche humana en polvo. Todas las cepas mostraron resistencia a los pH bajos, a las sales biliares y a la exposición a enzimas gástricas, así como una buena hidrofobicidad y capacidad de autoagregación. Además, no presentaron actividad de hemaglutinación o hemólisis en un medio de cultivo rico en eritrocitos. Observamos que el consumo de leche humana en polvo evita en los ratones el aumento de peso y el estreñimiento. Conclusiones: después del secado por aspersión, las cepas con posible potencial probiótico pueden conservarse en la leche materna. El consumo de leche humana en polvo con bacterias probióticas evita el estreñimiento y el aumento de peso en los ratones, en comparación con los alimentados con leche de una formula comercial.

Antimicrobial peptide presenting potential strain-specific real time polymerase chain reaction assay for detecting the probiotic Lactobacillus reuteri KUB-AC5 in chicken intestine.

The gene coding for antimicrobial peptides produced by probiotic Lactobacillus reuteri KUB-AC5 located on the cloned DNA fragment I-C46 containing 2 open reading frames I-C46-F2.1 and I-C46-F2.2 were designed for strain-specific primers P1 and P2, respectively, and assessed by real-time quantitative polymerase chain reaction. According to the obtained results, primer P1 has limited strain specificity. Primer P2 exhibited high efficacy and specificity at annealing temperature of 70°C while P1 annealed at 57°C causing nonspecific bands. Hence, P2 was selected for quantitative polymerase chain reaction assay by isothermal annealing and extension reaction at high temperature of 70°C resulting in linearity for its DNA sequences ranging from 102 to 107 target copy numbers per assay, and displaying a detection limit of 6.17 log cfu/g of cecal digesta. Using spike testing, this system was able to detect 7.88 ± 0.06 to 11.78 ± 0.06 log copy number/g of digesta, higher than cultivation assay at about 1 log cfu/g, with good correlation of 0.99. These results suggested possible detection of strain KUB-AC5 in the gastrointestinal tract of chicken to evaluate the efficacy and persistence of a probiotic strain which requires correct inclusion rates in the feed.

A Randomized Controlled Open Label Crossover Trial to Study Vaginal Colonization of Orally Administered Lactobacillus Reuteri RC-14 and Rhamnosus GR-1 in Pregnant Women at High Risk for Preterm Labor.

Lactobacilli administration has been suggested for the treatment and prevention of bacterial vaginosis, which increases the risk for preterm birth. We aimed to evaluate the vaginal colonization of lactobacilli orally administered to pregnant women at risk for preterm birth. We performed a randomized and controlled crossover study between January 2016 and May 2017. Forty pregnant women at high risk for preterm birth with normal vaginal flora (Nugent score ≤ 3) were randomized to either receive two oral capsules/day each containing 5 × 109 Lactobacilli (L.) rhamnosus GR-1 and L. reuteri RC-14 (n = 20) or no treatment (n = 20) for 2 months. Treatments were then crossed over for an additional two months. A vaginal examination and swabbing were performed for assessment of bacterial vaginosis at baseline and every month until study completion. At the same time points, vaginal samples were cultured and subjected to matrix-assisted-laser-desorption/ionization-time-of-flight-mass-spectrometry (MALDI TOF-MS) for the detection of the specific bacterial strains contained in the capsules. The primary endpoint was the presence of the administered lactobacilli strains in the vagina during the first two months of follow-up. Thirty-eight women completed the study. During the first two months of treatment, L. rhamnosus GR-1 was detected in one (5%) woman on the probiotic treatment and 2 (11%) women receiving no treatment (p = 0.6). L. rhamnosus GR-1 was detected in vaginal samples of 4 (11%) women during probiotic treatment (of both groups) and L. reuteri RC-14 was not detected in any samples. The rest of the endpoints were not different between the groups. Altogether, vaginal colonization of lactobacilli following oral administration is low during pregnancy.

Lactobacillus reuteri A9 and Lactobacillus mucosae A13 isolated from Chinese superlongevity people modulate lipid metabolism in a hypercholesterolemia rat model.

While there is strong evidence showing that many food-borne probiotics regulate cholesterol metabolism, few studies have examined how probiotics of human origin affect cholesterol metabolism. Because people living in so-called 'longevity villages' are unlikely to have hypercholesterolemia, we hypothesized that probiotics isolated from the residents would have cholesterol-reducing effects on rats with hypercholesterolemia. We isolated 16 strains of Lactobacillus from four longevity populations in China. The strains were tested in vitro for bile salt hydrolase (BSH) activity and two isolates, Lactobacillus reuteri A9 and Lactobacillus mucosae A13, were screened out. These two strains were then administered daily for 28 d to rats fed a cholesterol-rich diet. The serum total cholesterol levels in the L. reuteri A9 and L. mucosae A13 groups decreased by 24.3% and 21.6%, respectively. The serum low density lipoprotein cholesterol levels decreased by 23.8% and 25.2%, respectively. The L. reuteri A9 and L. mucosae A13 groups also exhibited upregulated hepatic mRNA expression of Sterol regulatory element-binding protein 2 (Srebp2) by 2.71-fold and 2.54-fold, respectively. The mRNA expression levels of hepatic low-density lipoprotein receptor (Ldlr) in the two groups were significantly up-regulated by 1.28-fold and 2.17-fold, respectively. The composition of gut microbiota was recovered by oral gavage in both experimental groups, and the destroyed diversity of gut microbiota was relieved.

Oral probiotics and the female urinary microbiome: a double-blinded randomized placebo-controlled trial.

PURPOSE: Probiotics may reduce risk of urinary tract infection by preventing colonization of uropathogens. We aimed to determine the change in the ratio between uropathogens:Lactobacillus (U/L) within the lower urinary tract in response to oral probiotic. METHODS: This was a double-blinded randomized controlled trial of healthy pre-menopausal female volunteers. Participants provided daily voided urine for 3 months including three phases of the trial: 1-baseline, 2-intervention, 3-wash-out. Participants were randomized to an oral probiotic (Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14) versus placebo. The primary outcome was the U/L ratio of daily voided urine, as determined by an enhanced urine culture method. Analysis included t test of the ratios and separate generalized linear mixed effects models (GLMM) for microbiota diversity. RESULTS: 481 samples of seven female participants with mean age 29.1 years (± 5.3 years) were included in the analysis (probiotic n = 4; placebo n = 3). No adverse events were reported. The placebo and probiotic groups had similar mean U/L ratios with no difference between placebo and probiotic groups in Phases 1-3 (p = 0.90, p = 0.58 and p = 0.72, respectively). The probiotic species were never identified in the voided urine. There were no changes between groups in terms of microbiota diversity. CONCLUSION: For young healthy women, the use of oral probiotic did not affect the U/L ratio.