Probiotic Lactobacillus Species Research Articles

β-galactosidase producing Lactiplantibacillus spp in intestinal microbiome mouse diarrhea model and metagenomic analyses

Probiotics have attained significant interest in recent years as a result of their gut microbiome modulation and gastrointestinal health benefits. Numerous fermented foods contain LAB with GRAS grade and probiotic bacteria. The most promising isolates with high potentiality were selected for 16S rRNA gene sequence analysis and identified as Lactiplantibacillus plantarum, L. fermentum, L. pentosus and Lactiplantibacillus sp. The present study was conducted to determine the gastrointestinal symptoms after the consumption of milk and dairy products, lactose intolerance status using mice model. Post-weaning Balb/c mice were orally administered with probiotic bacteria to assess their efficiency in reducing lactose intolerance symptoms. They exhibited significantly reduced total feces weight and slower intestinal motility and they efficiently alleviated diarrhea symptoms within 6 h of lactose challenge in lactose intolerance-induced mice. Next generation sequence analysis was performed using intestinal content of model treated with L. plantarum GV54 and GV64, the intestinal bacterial diversity was studied using Illumina NGS analysis (16S rRNA hypervariable regions [V3 and V4]). NGS report confirmed the existence of the dominant phyla of the gut microbiome in mice are Bacteroidetes, Firmicutes and Proteobacteria. Due to the influence of the probiotic bacteria in the model group (GV54 and GV64), maximum abundance of phylum Firmicutes were observed when compared to the control group, whereas control group showed more abundance of phylum Bacteriodetes. However, the relative abundance of Bacteroidetes in the model group was decreased and Firmicutes abundance was increased. This indicates the genus abundance of Lactobacillus (Lactiplantibacillus) species or Lactiplantibacillus plantarum GV54 and GV64 bacteria. After treatment with probiotic bacteria Lactobacillus (Lactiplantibacillus) species, the probiotic bacterial abundance was increased consequently genera Helicobacter, Clostridiales unclassified and Desulfovibrio were decreased in the model groups. Probiotic bacteria has a positive impact on the regulation of intestinal microbial diversity in the tested mice system and it reflects that the intestinal microbiota of mice is dynamic and has positive impact on gut health. Considering this point, it is possible to promote Lactiplantibacillus spp. as probiotics and therapeutic food development at industrial scale.

Identification of immune-associated genes with altered expression in the spleen of mice enriched with probiotic Lactobacillus species using rna-seq profiling.

Probiotics are living microorganisms that can provide health benefits when consumed. Here, we investigated the effects of probiotics on gene expression in the spleen of mice using RNA-sequencing analysis between negative control and probiotic groups (including 4 Lactobacillus strains: Lactobacillus fermentum, L. casei, L. plantarum, and L. brevis). Mice exposed with probiotic in 4 weeks by intragastric administration. Then, spleen tissues of the control and probiotics groups were collected on days 14 and 28 for RNA sequencing. In total, 665, 186, and 81 DEGs were significantly expressed on day 14 vs. control, day 28 vs. control groups, and probiotics day 28 vs. day 14 groups, respectively. On the other hand, 12 Toll-like receptor (TLR) genes underwent additional validation through qRT-PCR, affirming the increased alignment between qRT-PCR and RNA-Seq findings. In addition, the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses revealed that the DEGs were predominantly enriched in defense responses to pathogens, including inflammatory bowel diseases, malaria, leukaemia virus 1, and herpes virus, as well as immune processes related to immune response and signal transduction. This study represents the first investigation into mice's gene expression in the spleen exposed to probiotics using Lactobacillus spp. isolated from a field strain in Vietnam. Our results provide valuable insights into the impacts and functions of probiotics on mammalian development, offering crucial information for the potential therapeutic use of probiotics in defending against pathogens in Vietnam. The findings from this study highlight the potential of probiotics in modulating gene expression in the spleen, which may have implications for immune function and overall health in mice.

Role of Native Probiotic Lactobacillus Species via TGF-β Signaling Pathway Modulation in CRC

Colon microbiome composition in colorectal cancer (CRC) patients undergoes remarkable changes. The present study was designed to assess the impact of Lactobacillus mixture on the regulating the CRC by influencing the transforming growth factor beta (TGF-β) signaling pathway in both in vitro (HT-29 cancer cells) and in vivo (BALB/c mice) models. In this study, the antiproliferative effect of a native potential probiotic Lactobacillus mixture on HT-29 cancer cells was evaluated using the MTT assay method. Also, qRT-PCR was performed to assess the RNA expression level of genes associated with the TGF-β signaling pathway at three levels: receptor, regulatory, and inhibitory SMADs. Finally, the in vivo assays were investigated by three groups of mice: a naive group (PBS), a disease group (azoxymethane [AOM]/ dextran sulfate sodium [DSS] + PBS), and a treatment group (AOM/DSS + Lactobacillus mixture in PBS). The MTT results showed a significant decrease in proliferation of HT-29 cancer cells after 120 h of treatment. Furthermore, qRT-PCR demonstrated the downregulation of the smad2/3 gene expression in HT-29-treated cells and also reduction in the level of smad4 gene expression. In addition, in the mouse model, the tgf-βR1 gene was downregulated in the group treated with AOM/DSS/Lactobacillus, but not the AOM/DSS group. A downregulation of smad4 gene expression was also observed in in vivo models. The obtained results suggest that our novel probiotic Lactobacillus mixture could have a positive impact on the inhibition of the CRC progression by downregulating the TGF-β signaling pathway.

Molecular diagnosis for camel raw milk microbiota.

The existence of diverse microbes in unprocessed camel milk poses a significant threat to the well-being of a large population, especially infants and toddlers. The objective of this study was to ascertain the existence of microorganisms in unprocessed raw camel milk by employing a molecular-based technique in combination with a histological examination of bacteria. The identification of microbial species was achieved by employing PCR amplification and sequencing of 16s rRNA gene fragments. Various micorganisms found includes the probiotic Lactobacillus species, Staphylococcus succinic, Macrococcus casealyticus, Bacillus cohnii, and Salinicoccus kunmingensis. To prevent microbial contamination in raw milk, it is necessary to adequately heat or pasteurise the milk and to wash and sterilise the udder before milking the camel. This is because raw milk contains microbes that cause multiple diseases. Moreover, in the current era of the COVID-19 pandemics, ensuring proper sanitary conditions in milk and its derivatives might potentially mitigate the transmission of various diseases among consumers shortly. Keywords: camel, microbiota, 16s rRNA gene, PCR.

Dietary Lactobacillus johnsonii-derived extracellular vesicles ameliorate acute colitis by regulating gut microbiota and maintaining intestinal barrier homeostasis.

Inflammatory bowel disease (IBD) is a chronic gastrointestinal disease with intricate pathogenesis, and clinical treatment is still not ideal. The imbalance of gut microbiota is associated with IBD progression. Various probiotics have been used as functional foods for the prevention and treatment of IBD, but the specific mechanism is still not fully understood. Lactobacillus johnsonii (L. johnsonii) is a potential anti-inflammatory bacterium, and compared to other probiotic Lactobacillus species, its colonization in the gut of colitis patients is significantly reduced. In this study, we first found that dietary L. johnsonii exerts strong anti-inflammatory and antioxidant effects in colitis mice, and this beneficial effect is directly related to its derived extracellular vesicles (LJ-EVs). Further experimental results indicate that LJ-EVs effectively prevented colitis symptoms and modulated gut microbiota and metabolic pathways. Meanwhile, we have studied for the first time the protective effect of LJ-EVs on the intestinal barrier from the perspective of reducing oxidative stress. We found that LJ-EVs can be directly taken up by intestinal epithelial cells and activate the Nrf2/HO-1 antioxidant signaling pathway, reducing endotoxin damage to cells and maintaining intestinal barrier homeostasis, which cascades to alleviate intestinal inflammation response. This study reveals the mechanism of L. johnsonii in treating colitis and provides a new approach for the development of oral LJ-EVs for the treatment of colitis.

Human vaginal microbiota colonization is regulated by female sex hormones in a mouse model.

Clinically, a Lactobacillus rich vaginal microbiota (VMB) is considered optimal for reproductive outcomes, while a VMB populated by anaerobes is associated with dysbiosis and the clinical condition bacterial vaginosis (BV), which is linked to increased susceptibility to sexually transmitted infections and adverse reproductive outcomes. Mouse models that mimic eubiotic and dysbiotic VMB are currently lacking but could play a critical role in improving protective interventions. In this study, probiotic, eubiotic, and dysbiotic models were developed in C57BL/6 mice, using probiotic strains Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14, eubiotic Lactobacillus crispatus, or dysbiotic Gardnerella vaginalis strains. Endogenous sex hormones were manipulated by either ovariectomizing (OVX) mice or administering 17β-estradiol or progesterone pellets in OVX mice. Hormone-altered mice were inoculated with probiotic Lactobacillus species, L. crispatus, or G. vaginalis, and colonization was tracked using quantitative plating assays. Glycogen and MUC-1 levels in hormone-treated mice were determined with ELISA and MUC-1 staining. Following a single administration, L. rhamnosus and L. reuteri persisted in the mouse vaginal tract for up to eight days, L. crispatus persisted for up to three days, and G. vaginalis persisted for up to two days, as measured by quantitative plating assays and qPCR. Colonization of G. vaginalis was facilitated by the presence of mucin. The lack of endogenous hormones in OVX mice dramatically decreased VMB bacterial load compared to normal mice. None of the exogenous bacteria including Lactobacilli could colonize OVX mice for more than 24 hours. Treatment with 17β-estradiol but not progesterone restored the endogenous VMB and colonization with Lactobacilli and G. vaginalis. Interestingly, 17β-estradiol treated mice had significantly increased levels of glycogen compared to OVX and progesterone-treated mice. Based on the results, we have shown that estrogen played a significant role in the ability for human VMB species to colonize in our mouse models, potentially through a glycogen mediated mechanism. These results suggest there is a dynamic interaction between sex hormones and the VMB, which can affect bacterial diversity and the ability for a VMB to colonize.

Effects of probiotic Lactobacillus species on the kidney and liver tissues of type 2 diabetic mice

Effects of probiotic Lactobacillus species on the kidney and liver tissues of type 2 diabetic mice

Anti-Biofilm Potential of Lactobacillus casei and Lactobacillus rhamnosus Cell-Free Supernatant Extracts against Staphylococcus aureus.

Biofilm production is an important virulence factor in Staphylococcus aureus. Most of the infections associated with biofilms of this bacterium are very difficult to treat using antibiotics. The present research studied the effects of the two probiotic Lactobacillus species L. casei and L. rhamnosus on S. aureus biofilm. Cell-free supernatant (CFS) extracts of L. casei ATCC 39392 and L. rhamnosus ATCC 7469 culture were prepared. The effects of sub-minimum inhibitory concentrations of the CFS extracts on cell surface hydrophobicity (CSH), initial attachment, biofilm formation, and their ability in eradicating S. aureus ATCC 33591 biofilms were assessed. In addition, the effects of CFS extracts on expression of the genes involved in formation of S. aureus biofilms (cidA, hld, sarA, icaA, and icaR) were also evaluated through real-time polymerase chain reaction. CFSs of both Lactobacillus spp. significantly reduced CSH, initial attachment, and biofilm formation and eradicated the biofilms. The above findings were supported by scanning electron microscopy results. These two Lactobacillus CFSs significantly changed the expression of all studied biofilm-related genes. Expression levels of cidA, hld, and icaR genes significantly increased by 4.4, 2.3, and 4.76 fold, respectively, but sarA and icaA genes were significantly downregulated by 3.12 and 2.3 fold. The results indicated that CFS extracts of L. casei and L. rhamnosus had desirable antagonistic and anti-biofilm effects against S. aureus. Consequently, carrying out further research enables us to prepare pharmaceuticals from these CFSs in order to prevent and treat infections caused by S. aureus biofilms.

Tomato-fruit-derived extracellular vesicles inhibit Fusobacterium nucleatum via lipid-mediated mechanism.

Nano-sized extracellular vesicles (EV) are essential for cell communication. Studies on EV from natural sources including edible plants are gaining momentum due to the biological implications. In this study, EV from tomato fruit were isolated by ultracentrifugation and their physical and morphological features along with their biocargo profiles were analyzed. We found that tomato EV promote the growth of probiotic Lactobacillus species, while inhibiting growth of the opportunistic intestinal pathogens Clostridioides difficile and Fusobacterium nucleatum. Tomato EV reversed microbiota dysbiosis caused by F. nucleatum in a simulator of the gut microbiota fermentation model. Phospholipid analysis of tomato EV revealed that the anti-bacterial effect of tomato-EV was driven by the presence of specific lipids in the EV, as demonstrated by lipid depletion and reconstitution experiments. The findings suggest the potential of tomato-derived EV for treating gut microbiota dysbiosis and preventing intestinal bacterial infections.

Safety Evaluation by Phenotypic and Genomic Characterization of Four Lactobacilli Strains with Probiotic Properties.

Probiotic Lactobacillus species are known to exert health benefits in hosts when administered in adequate quantities. A systematic safety assessment of the strains must be performed before the Lactobacillus strains can be designated as probiotics for human consumption. In this study, we selected Lactobacillus fermentum IDCC 3901, L. gasseri IDCC 3101, L. helveticus IDCC 3801, and L. salivarius IDCC 3551 as representative Lactobacilli probiotic strains and investigated their probiotic properties and potential risks through phenotypic and genomic characterization. Various assays including antimicrobial resistance, biogenic amine production, L-/D-lactate production, acute oral toxicity, and antipathogenic effect were performed to evaluate the safety of the four Lactobacillus strains. Genomic analysis using whole genome sequencing was performed to investigate virulence and antibiotic resistance genes in the genomes of the selected probiotic strains. The phenotypes of the strains such as enzymatic activity and carbohydrate utilization were also investigated. As a result, antibiotic resistances of the four Lactobacillus species were detected; however, neither antibiotic resistance-related genes nor virulence genes were found by genomic analysis. Moreover, the four Lactobacillus species did not exhibit hemolytic activity or β-glucuronidase activity. The biogenic amine production and oral acute toxicity were not shown in the four Lactobacillus species, whereas they produced D-lactate with minor ratio. The four Lactobacillus species exhibited antipathogenic effect to five pathogenic microorganisms. This study provides a way to assess the potential risks of four different Lactobacillus species and validates the safety of all four strains as probiotics for human consumption.

Lactobacillus cell-free supernatant as a novel bioagent and biosurfactant against Pseudomonas aeruginosa in the prevention and treatment of orthopedic implant infection.

The hypothesis was that probiotic Lactobacillus species (spp.) or their cell-free supernatant (CFS) are effective in inhibiting (a) planktonic growth of Pseudomonas aeruginosa (PA), (b) its adhesion to a Ti6Al4V-alloy surface, and (c) in dispersing biofilm once formed. (a) A planktonic co-culture containing PA(104 colony-forming unit [CFU]/ml) was combined with either Lactobacillus acidophilus, Lactobacillus plantarum (LP), or Lactobacillus fermentum (LF) at a suspension of 104 (1:1) or 108 CFU/ml (1:2). Lactobacillus and PA CFUs were then quantified. (b) Ti-6Al-4V discs were inoculated with PA followed by supplementation with CFS and adherent PA quantified. (c) Biofilm covered discs were supplemented with Lactobacillus CFS and remaining PA activity quantified. Results showed that whole-cell cultures were ineffective in preventing PA growth; however, the addition of CFS resulted in a 99.99 ± 0.003% reduction in adherent PA in all Lactobacillus groups (p < .05 in all groups) with no viable PA growth measured in the LF and LP groups. Following PA biofilm formation, CFS resulted in a significant reduction in PA activity in all Lactobacillus groups (p ≤ .05 in all groups) with a 29.75 ± 15.98% increase measured in control samples. Supplementation with CFS demonstrated antiadhesive, antibiofilm, and toxic properties to PA.

Lactobacillus reuteri-derived extracellular vesicles maintain intestinal immune homeostasis against lipopolysaccharide-induced inflammatory responses\xa0in\xa0broilers

BackgroundLactobacillus reuteri strains are widely used as probiotics to prevent and treat inflammatory bowel disease by modulating the host’s immune system. However, the underlying mechanisms by which they communicate with the host have not been clearly understood. Bacterial extracellular vesicles (EVs) have been considered as important mediators of host-pathogen interactions, but their potential role in commensals-host crosstalk has not been widely studied. Here, we investigated the regulatory actions of EVs produced by L. reuteri BBC3, a gut-associated commensal bacterium of Black-Bone chicken, in the development of lipopolysaccharide (LPS)-induced intestinal inflammation in a chicken model using both in vivo and in vitro experiments.ResultsL. reuteri BBC3 produced nano-scale membrane vesicles with the size range of 60–250 nm. Biochemical and proteomic analyses showed that L. reuteri BBC3-derived EVs (LrEVs) carried DNA, RNA and several bioactive proteins previously described as mediators of other probiotics’ beneficial effects such as glucosyltransferase, serine protease and elongation factor Tu. In vivo broiler experiments showed that administration of LrEVs exerted similar effects as L. reuteri BBC3 in attenuating LPS-induced inflammation by improving growth performance, reducing mortality and decreasing intestinal injury. LrEVs suppressed the LPS-induced expression of pro-inflammatory genes (TNF-α, IL-1β, IL-6, IL-17 and IL-8), and improved the expression of anti-inflammatory genes (IL-10 and TGF-β) in the jejunum. LrEVs could be internalized by chicken macrophages. In vitro pretreatment with LrEVs reduced the gene expression of TNF-α, IL-1β and IL-6 by suppressing the NF-κB activity, and enhanced the gene expression of IL-10 and TGF-β in LPS-activated chicken macrophages. Additionally, LrEVs could inhibit Th1- and Th17-mediated inflammatory responses and enhance the immunoregulatory cells-mediated immunosuppression in splenic lymphocytes of LPS-challenged chickens through the activation of macrophages. Finally, we revealed that the reduced content of both vesicular proteins and nucleic acids attenuated the suppression of LrEVs on LPS-induced inflammatory responses in ex vivo experiments, suggesting that they are essential for the LrEVs-mediated immunoregulation.ConclusionsWe revealed that LrEVs participated in maintaining intestinal immune homeostasis against LPS-induced inflammatory responses in a chicken model. Our findings provide mechanistic insight into how commensal and probiotic Lactobacillus species modulate the host’s immune system in pathogens-induced inflammation.

Microbiological Studies on Naturally Present Bacteria in Camel and Buffalo Milk

The aim of current study was to isolate and identify naturally occurring probiotic Lactobacillus species in buffalo milk, camel milk, and camel urine to investigate their susceptibility to antibiotics and their antibacterial activity against pathogenic bacteria. A total number of seven samples which included three milk samples from buffalo, three milk samples from camel, and one urine sample from camel were collected and used in this study. The samples were cultured, and 18 isolated strains were identified by using 16S rRNA multiplex Polymerase Chain Reaction analysis, which was performed following DNA extraction from the isolated bacteria. Buffalo and camel milk were different in their Lactobacilli content. All Lactobacilli strains that were found in both camel milk and camel urine, were also found in buffalo milk, Lactobacilli strains in camel milk and urine were generally more resistant to the antibiotic. Lactobacilli isolated from buffalo milk, camel milk, and also camel urine presented variable degrees of antibacterial activity against pathogenic bacteria. Further studies should be conducted with more samples to gain more information in the field of antibacterial activity of probiotic lactobacilli and to understand the mechanisms of their activity. Hopefully, they can be used as natural alternatives instead of synthetic antibiotics.

Identification and Sensitivity of Vaginal and Probiotic Lactobacillus species to Urinary Antibiotics.

Objectives Healthy vaginal microbiota is mainly dominated by Lactobacillus species namely L. crispatus , L. gasseri , L. jensenii , and L. iners . Lactobacilli are thought to play an important role in the prevention of urogenital infections, and Lactobacillus probiotics to restore and/or maintain vaginal health has been advocated. These can interfere with the adherence, growth, and colonization by uropathogenic bacteria, thus reducing the risk of urinary tract infection (UTI). This study aims to isolate and evaluate the susceptibility of healthy vaginal and probiotic Lactobacillus spp. to urinary antibiotics. Materials and methods A total of 50 premenopausal, nonmenstruating women with no symptoms of vaginal infection or UTI or antimicrobial use in the past 2 weeks were enrolled. Two high vaginal swabs were collected for Nugent’s scoring and anaerobic culture. Colonies yielding gram-positive rods were confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Lactobacillus strains in a vaginal probiotic tablet were also isolated, and the sensitivity of both sets to urinary antibiotics was determined. Results A total of 29 Lactobacillus spp. were isolated, including L. crispatus (15), L. gasseri (5), L. vaginalis (4), L. paracasei (2), L. curvatus (1), L. fermentum (1), and L. paraplantarum (1). All strains were susceptible to nitrofurantoin and resistant to norfloxacin, gentamicin, and cotrimoxazole (except L. paracasei ). The probiotic strains were pan-sensitive. Conclusions Prophylactic antibiotics are capable of eliminating the normal vaginal inhabitants, which may increase the probability of UTI. The administration of vaginal probiotics as an alternate or multidrug therapy can restore vaginal microbiota and help prevent recurrent UTI.

Toxicity Analysis of Recombinant L-asparaginase I and II in Zebrafish.

L-asparaginases are extensively applied in the treatment of Acute Lymphoblastic Leukemia (ALL). The treatment regime of ALL consists of asparaginase from E. coli or Erwinia. The survival rate post-chemotherapy has increased to < 90% in recent years. Asparaginase therapy has also resulted in numerous toxicities to patients receiving therapy. This study demonstrates the reaction of normal cells of Danio rerio to asparaginase therapy. L-asparaginase I and II used in the present study are from two probiotic Lactobacillus species in comparison with a commercial L-asparaginase of E. coli origin. Zebrafish adults were injected with 2500 U/kg body weight of L-asparaginase treatments. The expression of SOD 2, CAT, GST, GTP BP3, FADS2 were analyzed with EF1α as house-keeping gene. The p value obtained proves that the data are significant. The histology of the L-asparaginase I treated fishes showed dilated sinusoids in the liver and pseudocyst in the pancreas. The L-asparaginase II and commercial asparaginase showed no pathology.

Isolation and identification of potential probiotic Lactobacillus species from feces of infants in southwest Iran

ObjectivesTo evaluate the potential probiotic properties of Lactobacillus strains isolated from feces of infants and also to determine their antimicrobial activity against some enteropathogenic bacteria. MethodsThe Fecal samples were prepared from 120 infants aged less than 24 months. In total, 105 Lactobacillus strains were identified by phenotypic tests. Thirty isolates were randomly selected to study their potential probiotic properties. These isolates were examined for resistance to acid (pH: 2.5, 2 h) and bile (oxgall 0.3%, 8 h), adhesion to HT-29 cells, antibiotic susceptibility, and antimicrobial activities. ResultsOn basis of 16S rRNA sequencing, 30 isolates identified as Lactobacillus fermentum (n = 11; 36.7%), Lactobacillus plantarum (n = 9; 30%), Lactobacillus rhamnosus (n = 6; 20%), and Lactobacillus paracasei (n = 4; 13.3%). All tested strains survived at acid and bile conditions. Six Lactobacillus strains revealed high adherence to HT-29 cells. Three strains including the L. fermentum (N2, N7), and the L. plantarum (N20) showed good probiotic potential and inhibited the growth of Yersinia enterocolitica ATCC 23715, Shigella flexneri ATCC 12022, Salmonella enterica ATCC 9270, and enteropathogenic Escherichia coli (EPEC) ATCC 43887. The antibiotic resistance test showed that all the isolates were susceptible to tetracycline, and chloramphenicol. ConclusionsLactobacillus strains like L. fermentum (N2, N7), and the L. plantarum (N20), could be potential probiotic, but further in vitro and in vivo studies on these probiotic strains are still required.

Lactobacillus species inhibitory effect on colorectal cancer progression through modulating the Wnt/β-catenin signaling pathway.

Probiotic bacteria are known to exert a wide range of anticancer activities on their animal hosts. In the present study, the anticancer effect of a cocktail of several potential probiotic Lactobacillus species (potential probiotic L.C) was investigated in vitro and in vivo. MTT and Flow cytometry tests results showed that administration of live potential probiotic L.C significantly decreased the HT-29 and CT-26 cells proliferation and induced late apoptotis in a time-dependent manner. In addition, quantitative real-time polymerase chain reaction (qPCR) results showed that exposure of potential probiotic L.C to both HT-29 and CT-26 cells during the incubation times resulted in the upregulation (apc and CSNK1ε for HT-29, CSNK1ε and gsk3β for CT-26) and downregulation (CTNNB1, CCND1, pygo2, axin2 and id2) of the Wnt/β- catenin pathway-related genes in a time-dependent manner. The significance of in vitro anticancer effect of potential probiotic L.C was further confirmed in an experimental tumor model. Data from the murine model of colorectal cancer (CRC) induced by Azoxymethane (AOM) and Dextran Sulfate Sodium (DSS) showed significantly alleviated inflammation and tumor development in AOM/DSS/L.C-injected mice compared to the AOM/DSS-injected mice. Tumor growth inhibition was accompanied by potential probiotic L.C-driven upregulation and downregulation of the Wnt/β-catenin pathway-related genes, similar to the in vitro results. These results showed that potential probiotic L.C inhibited the tumor growth, and that its anticancer activity was at least partially mediated through suppressing the Wnt/β-catenin pathway. Overall, the present study suggested that this probiotic could be used clinically as a supplement for CRC prevention and treatment.

Assessing the Prebiotic Potential of Taro (Colocasia esculenta) with Probiotic Lactobacillus Species in an in Vitro Human Digestion System (P20-022-19)

ObjectivesTaro (Colocasia escuknta), a culturally important staple food of the native Hawaiian diet, is high in fiber content, reaching 4.1 g/100 g. Prebiotics are carbohydrates that are indigestible by the digestive tract, which can selectively stimulate probiotic growth and/or activities in the colon. Due to taro’s high fiber content, it was hypothesized to have prebiotic potential. This study aimed to evaluate the effect of taro on the growth and adherence of probiotic Lactobacillus species in an in vitro human digestion system for the improvement of the human gut microbiome. MethodsFour probiotic Lactobacillus species, including L. acidophilus, L. paracasei, L. rhamnosus, and L. plantarum, were individually paired with 2% (w/v) taro. In addition to taro, 2% (w/v) glucose and inulin were used as controls. The pairings were subjected to an in vitro human digestive tract simulation of the mouth, stomach, and intestinal conditions to assess the fate of tested probiotics. Furthermore, an auto-agglutination assay was conducted to evaluate the effect of taro on self-agglutination of the individual probiotics. Lastly, the Caco-2 cell line was used to determine whether taro could influence the ability of tested probiotics to adhere to human intestinal epithelial cells. ResultsResults indicated that L. acidophilus, L. paracasei, and L. plantarum experienced greater growth in the simulated intestinal tract when paired with taro than with inulin or glucose. In addition, L. paracasei showed strong self-agglutination ability and had the greatest adherence percentage to Caco-2 cells. ConclusionsIn conclusion, taro is a strain-specific potential prebiotic that can be utilized as a dietary aid to modulate a healthy gut microbiota. Funding SourcesUSDA-ARS.USDA-NIFA HatchUniversity of Hawai’i Mānoa MahiMicrobes Program

Chinese liver fluke Clonorchis sinensis infection changes the gut microbiome and increases probiotic Lactobacillus in mice.

Chinese liver fluke Clonorchis sinensis changes the host's immune system. Recently, it has been reported that helminths including C. sinensis can ameliorate immune-related diseases such as allergy. In addition, recent studies showed that helminth infection can alleviate immune-mediated disorders by altering the gut microbiome. However, changes in the gut microbiome due to C. sinensis have not been reported yet. In this study, changes in the gut microbiome of C57BL/6 mice infected with C. sinensis metacercariae were evaluated over time. Stool was analyzed by 16S rRNA amplicon analysis using high-throughput sequencing technology. There was no apparent difference in species richness and diversity between the infected and control groups. However, the composition of the microbiome was different between the infected and control groups at 20days and 30days post-infection, and the difference disappeared at 50days post-infection. In particular, this microbiome alteration was associated with a change in the relative abundance of genus Lactobacillus and the probiotic Lactobacillus species that are known to have an immune-modulation role in immune-mediated diseases.

Broad range shuttle vector construction and promoter evaluation for the use of Lactobacillus plantarum WCFS1 as a microbial engineering platform.

As the field of synthetic biology grows, efforts to deploy complex genetic circuits in nonlaboratory strains of bacteria will continue to be a focus of research laboratories. Members of the Lactobacillus genus are good targets for synthetic biology research as several species are already used in many foods and as probiotics. Additionally, Lactobacilli offer a relatively safe vehicle for microbiological treatment of various health issues considering these commensals are often minor constituents of the gut microbial community and maintain allochthonous behavior. In order to generate a foundation for engineering, we developed a shuttle vector for subcloning in Escherichia coli and used it to characterize the transcriptional and translational activities of a number of promoters native to Lactobacillus plantarum WCFS1. Additionally, we demonstrated the use of this vector system in multiple Lactobacillus species, and provided examples of non-native promoter recognition by both L. plantarum and E. coli strains that might allow a shortcut assessment of circuit outputs. A variety of promoter activities were observed covering a range of protein expression levels peaking at various times throughout growth, and subsequent directed mutations were demonstrated and suggested to further increase the degree of output tuning. We believe these data show the potential for L. plantarum WCFS1 to be used as a nontraditional synthetic biology chassis and provide evidence that our system can be transitioned to other probiotic Lactobacillus species as well.