Assessment of probiotic strain Lactobacillus acidophilus LB supplementation as adjunctive management of attention-deficit hyperactivity disorder in children and adolescents: a randomized controlled clinical trial.

BACKGROUND: This study was designed to examine the possible efficacy of the probiotic strain Lactobacillus acidophilus LB (Lacteol Fort) on attention-deficit/hyperactivity disorder (ADHD) symptomatology and evaluate its influence on cognition function. METHODS: In this randomized controlled trial, 80 children and adolescents with ADHD diagnosis, aged 6-16 years, were included. The participants were randomly assigned to two groups: one group received probiotics plus atomoxetine, whereas the other group received atomoxetine only. ADHD symptomatology was assessed using the Conners Parent Rating Scale-Revised Long Version (CPRS-R-L) and Child Behavioral Checklist (CBCL/6-18). The participants were evaluated for their vigilance and executive function using Conner's Continuous Performance Test (CPT) and Wisconsin Card Sort Test (WCST). Both groups were assessed at the beginning of the study and the end of the twelve weeks. RESULTS: The probiotic group comprised 36 patients, whereas the control group comprised 40 patients in the final analysis after four patients dropped out of the trial. After 3 months of probiotic supplementation, a significant improvement in the CPRS-R-L and CBCL total T scores was observed compared with those in the control group (p = 0.032, 0.024, respectively). Additionally, the probiotic group demonstrated improved focus attention (target accuracy rate and omission errors;p = 0.02, 0.043, respectively) compared with the control group. An analysis of the Wisconsin Card Sorting Test (WCST) performance demonstrated that the probiotic group had significantly lower perseverative (p = 0.017) and non-perseverative errors (p = 0.044) but no significant differences compared to the control group. CONCLUSION: Lactobacillus acidophilus LB supplementation combined with atomoxetine for 3 months had a beneficial impact on ADHD symptomology and a favorable influence on cognitive performance. As a result, the efficacy of probiotics as an adjunctive treatment for managing ADHD may be promising. TRIAL REGISTRATION: ClinicalTrials.gov (identifier: NCT04167995). Registration date: 19-11-2019.

Positive efficacy of Lactiplantibacillus plantarum MH-301 as a postoperative adjunct to endoscopic sclerotherapy for internal hemorrhoids: a randomized, double-blind, placebo-controlled trial.

Background: Endoscopic sclerotherapy is a widely used minimally invasive procedure for internal hemorrhoids, yet postoperative symptoms remain a concern. The purpose of this study is to investigate the postoperative adjuvant efficacy of Lactiplantibacillus plantarum. Method: In this study, patients (≥18 years) with internal hemorrhoids that conformed to Goligher's classification of grade I-III received administration of L. plantarum MH-301 for 4 weeks following endoscopic sclerotherapy. The primary clinical endpoint in this study was the improvement rate, which was defined as the percentage of patients whose n-HDSS score decreased to 0 following the procedure. Stools were collected for high-throughput sequencing analysis post operation. Result: A total of 103 participants (51 in the LP group and 52 in the C group) were recruited, with 96 completing the entire trial (49 in the LP group and 47 in the C group). The primary clinical endpoint showed a higher improvement rate in the LP group (87.8% vs. 70.2%, P = 0.045). High-throughput sequencing analysis demonstrated that the LP group had a greater diversity of intestinal microbiota and a higher relative abundance of beneficial bacteria such as Bifidobacterium, Megamonas, and Lactobacillus. No significant difference in postoperative complications and adverse events was found. Conclusion: This paper concludes that the administration of L. plantarum MH-301 after endoscopic sclerotherapy can further increase the efficacy of the procedure and improve bowel movements. Regulation of intestinal microbiota may be the potential mechanism for the efficacy of L. plantarum MH-301.

Genetic and phenotypic assessment of the antimicrobial activity of three potential probiotic lactobacilli against human enteropathogenic bacteria.

Introduction: Lactobacilli are avid producers of antimicrobial compounds responsible for their adaptation and survival in microbe-rich matrices. The bactericidal or bacteriostatic ability of lactic acid bacteria (LAB) can be exploited for the identification of novel antimicrobial compounds to be incorporated in functional foodstuffs or pharmaceutical supplements. In this study, the antimicrobial and antibiofilm properties of Lactiplantibacillus pentosus L33, Lactiplantibacillus plantarum L125 and Lacticaseibacillus paracasei SP5, previously isolated form fermented products, were examined, against clinical isolates of Staphylococcus aureus, Salmonella enterica subsp. enterica serovar Enteritidis and Escherichia coli. Methods: The ability of viable cells to inhibit pathogen colonization on HT-29 cell monolayers, as well as their co-aggregation capacity, were examined utilizing the competitive exclusion assay. The antimicrobial activity of cell-free culture supernatants (CFCS) was determined against planktonic cells and biofilms, using microbiological assays, confocal microscopy, and gene expression analysis of biofilm formation-related genes. Furthermore, in vitro analysis was supplemented with in silico prediction of bacteriocin clusters and of other loci involved in antimicrobial activity. Results: The three lactobacilli were able to limit the viability of planktonic cells of S. aureus and E. coli in suspension. Greater inhibition of biofilm formation was recorded after co-incubation of S. enterica with the CFCS of Lc. paracasei SP5. Predictions based on sequence revealed the ability of strains to produce single or two-peptide Class II bacteriocins, presenting sequence and structural conservation with functional bacteriocins. Discussion: The efficiency of the potentially probiotic bacteria to elicit antimicrobial effects presented a strain- and pathogen-specific pattern. Future studies, utilizing multi-omic approaches, will focus on the structural and functional characterization of molecules involved in the recorded phenotypes.

[Assessment of the in vitro effect of intra and extracellular extracts of Lactobacillus against genotoxicity and oxidative stress caused by acrylamide]. / Evaluación del efecto in vitro de extractos intra y extracelulares de Lactobacillus contra la genotoxicidad y el estrés oxidativo causado por la acrilamida.

Introduction: Introduction: acrylamide is formed by the Maillard reaction and is found in many food products subjected to thermal processes, generating genotoxicity and DNA damage. Studies have reported that lactobacilli have the ability to generate compounds with antioxidant, antigenotoxic and antimutagenic activity, which is why the present work aims to evaluate the effect of Lactobacillus strains and their intra and extracellular extracts against genotoxicity and oxidative stress as caused by acrylamide. Methods: a strain of Lactobacillus casei Shirota and a strain of Lactobacillus reuteri NRRL B-14171 were used, both were cultured in MRS broth and subjected to mechanical and enzymatic treatments to obtain extra and intracellular extracts. Lymphocytes were cultured in RPMI medium. Lipid peroxidation was evaluated by TBARS and the antioxidant capacity was measured in the extra and intracellular extracts with the ABTS technique, also using a strain of Saccharomyces cerevisiae RC 212 as a model. The reduction of lipid peroxidation in lymphocytes was measured by TBARS and the reduction of genotoxicity by reducing the formation of micronuclei in lymphocytes. Results: both strains evaluated, as well as their intra and extracellular extracts, showed the ability to counteract oxidative stress and genotoxicity caused by acrylamide. Conclusion: the results found suggest that the use of intra and extracellular extracts of both strains could be an alternative to reduce the effects of genotoxicity and oxidative stress caused by acrylamide without the need for a viable structure.

Introducción: Introducción: la acrilamida se forma mediante la reacción de Maillard, por lo que se encuentra en muchos productos alimenticios sometidos a procesos térmicos, generando genotoxicidad y daños al ADN. Los estudios han reportado que los lactobacilos tienen la capacidad de generar compuestos con actividad antioxidante, antigenotóxica y antimutagénica, y es por esto que el presente trabajo pretende evaluar el efecto de cepas de Lactobacillus y sus extractos intra y extracelulares contra la genotoxicidad y el estrés oxidativo causado por la acrilamida. Métodos: se empleó una cepa de Lactobacillus casei Shirota y una cepa de Lactobacillus reuteri NRRL B-14171. Ambas fueron cultivadas en caldo MRS y sometidas a tratamientos mecánicos y enzimáticos para obtener los extractos extra e intracelulares. Los linfocitos fueron cultivados en medio RPMI, la peroxidación lipídica se evaluó por TBARS y la capacidad antioxidante se midió en los extractos extra e intracelulares con la técnica ABTS, empleando además una cepa de Saccharomyces cerevisiae RC 212 como modelo. La reducción de la peroxidación lipídica en los linfocitos se midió por TBARS y la reducción de la genotoxicidad mediante la reducción de la formación de micronúcleos en los linfocitos. Resultados: ambas cepas evaluadas, así como sus extractos intra y extracelulares, mostraron capacidad de contrarrestar el estrés oxidativo y la genotoxicidad causada por la acrilamida. Conclusión: los resultados encontrados, sugieren que el empleo de extractos intra y extracelulares de ambas cepas podría ser una alternativa para reducir los efectos de genotoxicidad y estrés oxidativo causados por la acrilamida sin la necesidad de requerir una estructura viable.

In Vitro Assessment of Lactobacillus crispatus UBLCp01, Lactobacillus gasseri UBLG36, and Lactobacillus johnsonii UBLJ01 as a Potential Vaginal Probiotic Candidate.

In this study, Lactobacillus crispatus UBLCp01, Lactobacillus gasseri UBLG36, and Lactobacillus johnsonii UBLJ01 isolated from the vagina of healthy reproductive age Indian women were screened for beneficial probiotic properties. These strains showed the ability to survive acidic and simulated vaginal fluid conditions and could adhere to mucin. Lact. gasseri UBLG36, and Lact. johnsonii UBLJ01 produced D- and L-lactic acid, whereas Lact. crispatus UBLCp01 produced hydrogen peroxide and D- and L-lactic acid. All strains inhibited the growth of pathogens (Escherichia coli, Gardnerella vaginalis, Proteus mirabilis, and Candida albicans) and were capable of co-aggregating with them with varying degrees. Strains secreted exopolysaccharides and formed biofilms under in vitro conditions. Safety assessment showed that these strains had a usual antibiotic susceptibility profile, did not produce hemolysins, gelatinases, and mucin degrading enzymes. Based on strain characteristics and beneficial properties, we believe that these strains are promising candidates for human trials to confirm their ability to prevent/treat vaginal dysbiosis and maintain a healthy vaginal eco-system.

Assessment of the Performance of the Aptima Bacterial Vaginosis Assay Over a 3-Month Period in a French Hospital.

Bacterial vaginosis (BV) is the most common cause of abnormal vaginal discharge. BV represents a dysbiosis with the acquisition of a diverse community of anaerobic bacteria and a reduction in lactobacilli burden. Our objective was to evaluate the Aptima BV assay kit for the diagnosis of BV. From May to August 2019, we enrolled outpatients and inpatients, including nonpregnant women above 18 with vaginosis symptoms, consulting at Nantes University hospital. The Aptima BV assay measures the loads of Gardnerella vaginalis, Atopobium vaginae, and Lactobacillus species in relation to overall bacterial load. The Aptima BV assay was compared to Nugent scoring (NS). A total of 456 women were enrolled, and 347 patients met the inclusion criteria with data available for the analysis. NS was used to classify the samples and 144 (41.5%) samples were classified as normal (NS = 0-3), 45 (13%) as BV (NS = 7-10), 38 (11%) presented an intermediate vaginal microbiota (3 < NS < 7), 79 (22.7%) had various bacteria (excluding vaginal flora), 29 (8.3%) had insufficient bacterial density, and 12 (3.5%) had a predominance of yeasts. The Aptima BV kit displayed a sensitivity of 91.1% and specificity of 94.4% with a positive predictive value (PPV) of 83.7% and a negative predictive (NPV) value of 97.1%. The results of this monocentric retrospective study show that Aptima BV kit has a good diagnostic correlation compared to standard of care for dysbiotic diagnosis cases. IMPORTANCE The possibility exists of the involvement of a new molecular test in the routine algorithm of bacterial vaginosis diagnosis in microbiology laboratories. This manuscript reports on our experience, and we propose an organization combining Nugent scoring and molecular testing, especially for intermediate Nugent scores.

MOLECULAR ASSESSMENT OF FECAL LACTOBACILLI POPULATIONS IN CHILDREN WITH FUNCTIONAL CONSTIPATION.

BACKGROUND: Investigation of the gut-specific bacterial strains including lactobacilli is essential for understanding the bacterial etiology of constipation. OBJECTIVE: This study aimed to compare the prevalence and quantity of intestinal lactobacilli in constipated children and healthy controls. METHODS: Forty children fulfilling Rome IV criteria for functional constipation and 40 healthy controls were recruited. Fecal samples were analyzed using species-specific polymerase chain reaction followed by random amplified polymorphic DNA-PCR and quantitative real-time PCR. RESULTS: Totally, seven different species of lactobacilli were detected. Out of 80 volunteers, 65 (81.3%) were culture and species-specific PCR positive from which 25 (38.46%) constipated children and 40 (61.54%) healthy subjects. The most prevalent species were L. paracasei 21 (32.3%) followed by L. plantarum 18 (27.7%) among both healthy and patient groups. Analysis of the RAPD dendrograms displayed that strains isolated from constipated and non-constipated children have similarity coefficients of more than 90%. The qPCR assays demonstrated constipated children had a lower amount of total lactobacilli population (per gram of feces) than healthy controls. CONCLUSION: Our findings showed that the mere existence of various species of Lactobacillus in the gut does not enough to prevent some gastrointestinal disorders such as functional constipation, and their quantity plays a more important role.

Assessment of dental caries lesion activity status using quantitative parameters obtained from the quantitative light-induced fluorescence method and difference of microbial distribution in primary molars.

BACKGROUND: This study aimed to assess differences in quantitative measures obtained from the quantitative light-induced fluorescence method and microbial composition of carious dentin and saliva according to the activity status of caries lesions in primary molars. METHODS: A total of 34 teeth from 34 children were evaluated in this study. The activity status of carious lesions was classified using the International Caries Classification and Management System criteria (active or inactive). Images of the carious lesions were captured using a quantitative light-induced fluorescence device for quantitative analyses. Carious dentin and saliva were collected to detect and quantify selected bacterial species (S. mutans, S. sobrinus, Lactobacillus species, F. nucleatum, P. nigrescence, P. intermedia) and C. albicans by quantitative polymerase chain reaction. Mann-Whitney U tests were performed to evaluate differences in quantitative measures from quantitative light-induced fluorescence, the microbial composition of carious dentin, and saliva according to the activity status of carious lesions. RESULTS: Red fluorescence values (∆R, ∆Rmax) from the quantitative light-induced fluorescence method were significantly higher in active lesions (∆R, p = 0.009; ∆Rmax, p = 0.014). The quantitative mean levels of Lactobacillus species (p = 0.010) in carious dentin and S. sobrinus (p = 0.017) in saliva were significantly higher in the active-lesion group. CONCLUSIONS: Quantitative measures related to red fluorescence from the quantitative light-induced fluorescence method, levels of Lactobacillus species from carious dentin, and levels of S. sobrinus from saliva were associated with caries lesion activity.

A Comparative Genomic and Safety Assessment of Six Lactiplantibacillus plantarum subsp. argentoratensis Strains Isolated from Spontaneously Fermented Greek Wheat Sourdoughs for Potential Biotechnological Application.

The comparative genome analysis of six Lactiplantibacillus plantarum subsp. argentoratensis strains previously isolated from spontaneously fermented Greek wheat sourdoughs is presented. Genomic attributes related to food safety have been studied according to the European Food Safety Authority (EFSA) suggestions for the use of lactic acid bacteria (LAB) in the production of foods. Bioinformatic analysis revealed a complete set of genes for maltose, sucrose, glucose, and fructose fermentation; conversion of fructose to mannitol; folate and riboflavin biosynthesis; acetoin production; conversion of citrate to oxaloacetate; and the ability to produce antimicrobial compounds (plantaricins). Pathogenic factors were absent but some antibiotic resistance genes were detected. CRISPR and cas genes were present as well as various mobile genetic elements (MGEs) such as plasmids, prophages, and insertion sequences. The production of biogenic amines by these strains was not possible due to the absence of key genes in their genome except lysine decarboxylase associated with cadaverine; however, potential degradation of these substances was identified due to the presence of a blue copper oxidase precursor and a multicopper oxidase protein family. Finally, comparative genomics and pan-genome analysis showed genetic differences between the strains (e.g., variable pln locus), and it facilitated the identification of various phenotypic and probiotic-related properties.

In Vitro assessment of anti-Campylobacter activity of lactobacillus strains isolated from canine rectal swabs.

BACKGROUND: Campylobacteriosis is currently the most frequently reported zoonosis. Dogs, especially puppies or those with diarrhea, are considered a possible source of human infection. Probiotic bacteria, such as Lactobacillus species, seem to be a valuable tool in controlling of intestinal pathogenic microorganisms in dogs. The main purpose of this study was to assess the anti-Campylobacter activity and some probiotic properties, like ability to produce H2O2, bile salt and low pH tolerance of Lactobacillus strains isolated from gastrointestinal tract of healthy dogs. RESULTS: A total of 39 rectal swabs derived from healthy dogs and 19 from dogs with diarrhea were examined to detect Lactobacillus and Campylobacter bacteria respectively. In total, 30 strains of Lactobacillus genus and four strains of Campylobacter genus were isolated and identified. Of the 30 strains of Lactobacillus, 22 showed an inhibitory effect towards Campylobacter. Four strains with the strongest antagonism towards Campylobacter bacteria (L. salivarius 25 K/L/1, L. rhamnosus 42 K/L/2, L. sakei 50 K/L/1 and L. agilis 55 K/L/1) were selected to assess their potential probiotic traits. Three out of four analyzed strains produced extracellular H2O2. All displayed very good or moderate survival at pH 3.0 and 2.0 and showed high tolerance to 0.5% and 1% bile salts. CONCLUSIONS: Among selected Lactobacillus strains, all may have a potential probiotic application in reducing Campylobacter spp. in dogs and thus prevent transmission of infection to humans, although the best candidate for probiotic seems to be L. sakei 50 K/L/1. Further in vitro and in vivo studies are needed.

Assessment of the microbiological origin of blowing defects in Grana Padano Protected Designation of Origin cheese.

Recognized worldwide for its history, flavor, and high nutritional quality, Grana Padano (GP) is one of the most traditional Italian raw-milk, hard-cooked, long-ripened cheese. Throughout GP manufacturing, some well-known and undesired bacterial species, such as clostridia, can proliferate and lead to spoilage defects that mischaracterize the final product; however, little is known about the development of late-blowing defects in hard cheese samples without clostridia. Therefore, in this study we aimed to use metataxonomic analysis to identify bacterial taxa associated with the development of late-blowing defect in GP samples. Furthermore, the presence of several heterofermentative lactobacilli species in defective zones were verified by primer-specific PCR assay. Considering α- and ß-diversity analyses, no statistically significant differences were detected between cheese samples with and without blowing defect. Following taxonomic assignment, Lactobacillus and Streptococcus were the dominant genera, whereas clostridia-related taxa were not detected in any of the 20 analyzed samples. Using EdgeR, the genera Propionibacterium and Acinetobacter were found to be prevalently more abundant in samples categorized as having "big regular holes." In samples with "small regular holes," multiplex PCR amplification revealed differences in terms of Lactobacillus population composition, mainly obligate homofermentative lactobacilli, between defective and non-defective zones of the same cheese wheel. This study demonstrated that GP samples with blowing defects not caused by clostridial development share similar biodiversity indices with GP collected from control zones, but an imbalance of obligate homofermentative lactobacilli was noticed between samples, which requires further analysis to better comprehend the exact mechanism involved in this process.

Clinical assessment and cytokines level in constipation-predominant irritable bowel syndrome participants treated with Lactobacillus-containing cultured milk drink.

BACKGROUND: Gut dysbiosis is linked with the pathophysiology of irritable bowel syndrome (IBS). Manipulation of intestinal microbiota using cultured milk drinks may stimulate the immune system, hence providing beneficial support in IBS treatment. This study aimed to investigate the effects of cultured milk drink on clinical symptoms, intestinal transit time (ITT), fecal pH and cytokines in constipation-predominant IBS (IBS-C) as compared to non-IBS participants. METHODS: Each recruited participant was given three bottles of 125 ml cultured milk drink containing 109 cfu Lactobacillus acidophilus LA-5 and Lactobacillus paracasei L. CASEI-01 consumed daily for 30 days. At pre- and post-30-day consumption, fecal pH, ITT, clinical symptoms, IL-6, IL-8 and TNF-α levels were assessed. Seventy-seven IBS-C and 88 non-IBS were enrolled. RESULTS: Post-consumption, 97.4% of IBS-C experienced improvements in constipation-related symptoms supported by the significant reduction of ITT and decreased fecal pH (p<0.05). All pro-inflammatory cytokines were significantly lower in post as compared to pre-consumption of cultured milk drinks in IBS-C (p<0.05). There was significant reduction in the IL-8 and TNF-α levels in post- as compared to pre-consumption for the non-IBS (p<0.05). CONCLUSION: Cultured milk drink taken daily improved clinical symptoms and reduced cytokines, hence should be considered as an adjunctive treatment in IBS-C individuals.

Assessment of probiotic adhesion and inhibitory effect on Escherichia coli and Salmonella adhesion.

In this study, we screened bacterial strains to identify specific probiotics to treat pig diarrhea caused by Escherichia coli or Salmonella. The potential probiotics were assayed for their survival in gastrointestinal solution, their antimicrobial activity, cell-surface properties, adhesion to Caco-2 cells, and inhibition of pathogen adhesion. Nine out of the 20 strains tested showed high tolerance of a simulated gastrointestinal environment and six strains exerted antagonistic effects against enterohemorrhagic E. coli (EHEC) O157:H7 and Salmonella Typhimurium MQ. Lactobacillus johnsonii pDX1e exhibited a higher potent antibacterial activity. Four strains (pDX1a, pDX1e, pDX3a, and pDX5a) displayed auto-aggregation, hydrophobicity, and adhesion to Caco-2 cells similar to those of the reference strain Lacticaseibacillus rhamnosus GG (LGG). Enterococcus durans pDX5a showed the highest adhesion capacity (13.86%), followed by the reference strain LGG (11.20%). All the tested strains competitively suppressed the attachment of pathogens to Caco-2 cells (by 30.73-55.18%); L. johnsonii pDX1e and Ent. durans pDX5a significantly inhibited the adhesion of pathogens by substitution and exclusion, respectively. Therefore, pDX1e and pDX5a were selected as probiotic strains for further investigation and application.

Systematic assessment of oat ß-glucan catabolism during in vitro digestion and fermentation.

ß-Glucan as a component of grain cell walls is consumed daily. However, little is known about whether ß-glucan is influenced by the gastrointestinal environment. In this study, we aim to investigate the integrated metabolic process of cereal ß-glucan. In vitro simulated digestion and fermentation combined with microbiome and metabolome analysis were used to profile the metabolism of ß-glucan. Intriguingly, we found that ß-glucan was not hydrolyzed by digestive enzymes but partially degraded by gastric acid environment during in vitro digestion. Moreover, ß-glucan was utilized by gut microbiota to produce acetate, propionate and butyrate, concurrently, the relative abundance of Lactobacillus significantly increased and Escherichia-Shigella significantly decreased. The correlation analysis between metabolomics datasets and microorganisms revealed that ß-glucan catabolism was also accompanied by amino acid catabolism and linoleic acid biosynthesis. Our study offered a forceful basis for the further exploration of the role of ß-glucan and gut microbiota in host health.

Ethanol tolerance assessment in recombinant E. coli of ethanol responsive genes from Lactobacillus buchneri NRRL B-30929.

We previously identified specific proteins associated with ethanol stress response in a Lactobacillus buchneri strain capable of growing in 10% ethanol. In the current study, the exceptional roles of ethanol responsive genes are examined to determine if they can increase ethanol tolerance in E. coli host cells. The recombinant strains carrying ethanol responsive genes were subjected to growth analyses in media with and without 4% ethanol. Among the expression of these genes and growth analyses of the recombinant strains in ethanol, six genes Lbuc_0522 (NADPH-dependent methylglyoxal reductase), Lbuc_0354 (succinate semialdehyde dehydrogenase), Lbuc_1211(threonyl_tRNA synthetase), Lbuc_2051 (nitroreductase), Lbuc_0707 (branched chain amino acid aminotransferase) and Lbuc_1852 (proline-specific peptidase) conferred host cells tolerance to 4% ethanol. Six genes Lbuc_1523 (phage major capsid protein, HK 97 family), Lbuc_1319 (phosphoglycerate kinase), Lbuc_0787 encoding fumarylacetoacetate hydrolase, Lbuc_1219 encoding UDP-N-acetylmuramate-L-alanine ligase, Lbuc_0466 encoding ornithine carbamoyltransferase and Lbuc_0858 encoding glycine hydroxymethyltransferase showed no impact on growth in media with 4% ethanol with IPTG induction when compared with E. coli carrying control pET28b plasmid. The expression of two genes Lbuc_1557 (S-layer glycoprotein) and Lbuc_2157 (6-phosphogluconate dehydrogenase) resulted ethanol sensitivity phenotype.

Assessment of commercial companion animal kefir products for label accuracy of microbial composition and quantity.

Kefir is a fermented beverage containing yeast and bacteria produced by the fermentation of water or milk with kefir grains. Lack of regulation for probiotic-containing fermented food sold for companion dogs and cats creates the potential for misreporting on viable microbial counts, taxonomy, and label claims. In this study, the microbiota of six companion animal kefir products were measured quantitatively using standard plating techniques. Microbial composition of these products was also characterized by using high-resolution, long-read amplicon sequencing of the 16S rRNA gene. Five products (83%) listed specific microorganisms, and four products (66%) guaranteed colony forming units (CFU)/g on their label. To enumerate viable lactic acid bacteria (LAB), two lots of each homogenized product were plated upon opening and following 14 d on deMan Rogosa and Sharpe (MRS) agar and incubated under anaerobic and aerobic conditions. Results from point of opening revealed that all commercial kefir products with a guaranteed CFU/g overstated the number of microorganisms present by at least 1 log, with only one product exceeding 1 × 109 CFU/g. Sequencing results demonstrated that none of the labels claiming specific bacterial genera and species on their labels were correct, and all products contained at least three additional bacterial species above the minimum detectable threshold (0.001% relative abundance) that were not disclosed by the manufacturer. In addition to the incorrect viable CFU and bacterial taxonomies, several of the product labels and websites contained a wide range of health claims, none of which are supported by the companion animal literature. Our results demonstrate a low level of accuracy in the labeling of commercial kefir products intended for use in dogs and cats. Regulatory agencies, veterinarians, pet food professionals, and pet owners must scrutinize these products and demand a higher level of accuracy and quality in the future.

Assessment of Techno-Functional and Nutraceutical Potential of Tomato (Solanum lycopersicum) Seed Meal.

Tomato (Solanum lycopersicum) is a widely consumed fruit all around the world. The industrial exploitation of tomato generates a lot of waste. Most of the utilization of tomato seeds waste is focused on animal feeding, as well as a food ingredient aimed to increase the protein content, and raw material for some organic bioactive component extraction. The aim of this work was to evaluate the techno-functional properties of tomato seed meal (TSM) and its nutraceutical properties after applying defatting processing (TSMD), and to evaluate the nutraceutical properties after a fermentation processing (TSMDF) by Lactobacillus sp. The results showed that, at alkaline conditions (pH 8-9), the techno-functional properties for TSM and TSMD improved. In comparison with TSM, TSMD showed higher water holding capacity (WHC ≈32%), higher oil holding capacity (OHC ≈13%), higher protein solubility (49-58%), more than 10 times foaming activity (FA), more than 50 times foam stability (Fst), as well as an improved emulsifying activity (EA) and emulsion stability (Est) wich were better at pH 9. Regarding the nutraceutical properties, after 48 h of fermentation (TSMDF), the antioxidant activity was doubled and a significant increase in the iron chelating activity was also observed. During the same fermentation time, the highest angiotensin-converting enzyme inhibition (ACEI) was achieved (IC50 73.6 µg/mL), more than 10 times higher than TSMD, which leads to suggest that this fermented medium may be a powerful antihypertensive. Therefore, the strategy proposed in this study could be an option for the exploitation of tomato wastes.

Probiotic potential and safety assessment of Lactobacillus isolated from yaks.

Current problem of antibiotic resistance and the high incidence of bacterial diseases has brought huge losses to the yak breeding industry in Tibet. Therefore, the purpose of this study was to isolate Lactobacillus with safety and beneficial probiotic potential for the prophylaxis of intestinal diseases in yaks. After 16S rDNA sequence, four strains i.e. Lactobacillus sakei (named L4), Enterococcus hirae (named E5), Pediococcus acidilactici (named P7), Weissella confusa (named W8) were isolated from feces of yaks. The results of tolerance to acid, bile salt, enzyme and temperature showed that P7 was highly tolerant to acid, bile salt and digestive enzyme, while E5 was more resistant to temperature. The antibacterial assay showed L4 had a strong inhibitory effect against Staphylococcus aureus (BNCC186335), and E5, P7, W8 had effective antibacterial ability against Escherichia coli (C83902). In addition, L4, E5, P7 and W8 mainly produced organic acids and bacteriocin production to inhibit common intestinal pathogens. The results of antibiotic susceptibility assay indicated that L4, E5, P7 and W8 were highly sensitive to most clinically used antibiotics and didn't contain the VanA and VanB genes on the basis of PCR amplification, and L4, E5, P7 and W8 didn't exhibit hemolytic activity. The animal toxicity experiment results showed that no obvious pathological change was found in intestinal tissue sections, and L4, E5 and W8 strains also promoted the growth performance of mice, consequently, the L4, E5, P7 and W8 had no toxic effect on mice. In conclusion, lactobacillus isolated from feces of yaks not only have potential probiotics and strong antibacterial ability in vitro, but also are safe. Therefore, they have the potential to reduce the occurrence of bacterial diseases as new feed additives.

Rapid in Vitro Assessment of Clostridioides difficile Inhibition by Probiotics Using Dielectrophoresis to Quantify Cell Structure Alterations.

Clostridioides difficile (C. difficile) infection (CDI) is the primary cause of nosocomial antibiotic-associated diarrhea, with high recurrence rates following initial antibiotic treatment regimens. Restoration of the host gut microbiome through probiotic therapy is under investigation to reduce recurrence. Current in vitro methods to assess C. difficile deactivation by probiotic microorganisms are based on C. difficile growth inhibition, but the cumbersome and time-consuming nature of the assay limits the number of assessed permutations. Phenotypic alterations to the C. difficile cellular structure upon interaction with probiotics can potentially enable rapid assessment of the inhibition without the need for extended culture. Because supernatants from cultures of commensal microbiota reflect the complex metabolite milieu that deactivates C. difficile, we explore coculture of C. difficile with an optimal dose of supernatants from probiotic culture to speed growth inhibition assays and enable correlation with alterations to its prolate ellipsoidal structure. Based on sensitivity of electrical polarizability to C. difficile cell shape and subcellular structure, we show that the inhibitory effect of Lactobacillus spp. supernatants on C. difficile can be determined based on the positive dielectrophoresis level within just 1 h of culture using a highly toxigenic strain and a clinical isolate, whereas optical and growth inhibition measurements require far greater culture time. We envision application of this in vitro coculture model, in conjunction with dielectrophoresis, to rapidly screen for potential probiotic combinations for the treatment of recurrent CDI.

Protease Amplification of the Inflammatory Response Induced by Commensal Bacteria: Implications for Racial Disparity in Term and Preterm Birth.

Decidual macrophages secrete proteases that activate protease-activated receptor 1 (PAR-1). We hypothesized that activation of the inflammatory response by bacteria is amplified by proteases, initiating labor. In addition, we hypothesized that commensal bacteria trigger an inflammatory response by activating NF-κB and TET methylcytosine dioxygenase 2 (TET2), a DNA de-methylase, via a protease amplified PAR-1, RhoA kinase (ROCK) pathway. To evaluate these hypotheses, we compared responses of mononuclear cells with Lactobacillus crispatus, prevalent in the vaginal microbiome of women of European ancestry, with L. iners and Fusobacterium nucleatum, which are more prevalent in vaginal samples collected from African-American women. Decidual tissue was collected at term not-in-labor (TNL), term labor (TL), spontaneous preterm labor (sPTL), and infected preterm labor (iPTL) and immunostained for PAR-1, TET2, and CD14. Mononuclear cells and THP-1 macrophage cells were treated with bacteria and elastase, a known activator of PAR-1. The inflammatory response was monitored by confocal microscopy of TET2 and the p65 subunit of NF-κB, as well as IL-8 production. Decidual staining for PAR-1, TET2, and CD14 increased TNL < TL < sPTL < iPTL. All treatments stimulated translocation of TET2 and p65 from the cytosol to the nucleus and increased IL-8, but L. iners and F. nucleatum caused more robust responses than L. crispatus. Inhibition of PAR-1 or ROCK prevented TET2 and p65 nuclear translocalization and increases in IL-8. Our findings demonstrate that proteases amplify the inflammatory response to commensal bacteria. The more robust response to bacteria prevalent in African-American women may contribute to racial disparities in preterm birth.