Strategies for the Identification and Assessment of Bacterial Strains with Specific Probiotic Traits.

Early in the 1900s, it was proposed that health could be improved and senility delayed by manipulating gut microbiota with the host-friendly bacteria found in yogurt. Later, in 1990, the medical community reconsidered this idea and today probiotics represent a developed area of research with a billion-dollar global industry. As a result, in recent decades, increased attention has been paid to the isolation and characterization of novel probiotic bacteria from fermented foods and dairy products. Most of the identified probiotic strains belong to the lactic acid bacteria group and the genus Bifidobacterium. However, current molecular-based knowledge has allowed the identification and culture of obligatory anaerobic commensal bacteria from the human gut, such as Akkermansia spp. and Faecalibacterium spp., among other human symbionts. We are aware that the identification of new strains of these species does not guarantee their probiotic effects and that each effect must be proved through in vitro and in vivo preclinical studies before clinical trials (before even considering it as a probiotic strain). In most cases, the identification and characterization of new probiotic strain candidates may lack the appropriate set of in vitro experiments allowing the next assessment steps. Here, we address some innovative strategies reported in the literature as alternatives to classical characterization: (i) identification of alternatives using whole-metagenome shotgun sequencing, metabolomics, and multi-omics analysis; and (ii) probiotic characterization based on molecular effectors and/or traits to target specific diseases (i.e., inflammatory bowel diseases, colorectal cancer, allergies, among others).

Role of Gut Microbiota and Probiotics in Colorectal Cancer: Onset and Progression.

The gut microbiota plays an important role in maintaining homeostasis in the human body, and the disruption of these communities can lead to compromised host health and the onset of disease. Current research on probiotics is quite promising and, in particular, these microorganisms have demonstrated their potential for use as adjuvants for the treatment of colorectal cancer. This review addresses the possible applications of probiotics, postbiotics, synbiotics, and next-generation probiotics in colorectal cancer research.

Antioxidant and Anti-Inflammatory Properties of Probiotic Candidate Strains Isolated during Fermentation of Agave (Agave angustifolia Haw).

Agave species are a source of diverse products for human use, such as food, fiber, and beverages, which include mezcal, a distilled beverage produced by spontaneous fermentation. Agave is an excellent source of high amounts of sugars, minerals, and phenolic compounds, which favor the growth of lactic acid bacteria (LAB) and yeast communities. In this work, 20 promising LAB strains with probiotic characteristics were isolated from the agave fermentation stage in mezcal production. The strains belonged to Lactobacillus plantarum (15), Lactobacillus rhamnosus (2), Enterococcus faecium (2), and Lactococcus lactis (1). These isolates were characterized for their resistance under gastrointestinal conditions, such as lysozyme, acid pH, and bile salts. In addition, the adherence of these LABs to human intestinal epithelial cells (Caco-2 and HT-29 cells) was tested in vitro and their antioxidant and immunomodulatory profile was determined using cellular models. Lactobacillus rhamnosus LM07 and Lactobacillus plantarum LM17 and LM19 strains were selected for their antioxidant properties, and their capacities in an oxidative stress model in intestinal epithelial cells IECs (Caco-2 and HT-29 cells) in the presence of hydrogen peroxide were evaluated. Interestingly, Lactobacillus rhamnosus LM07 and Lactobacillus plantarum LM17 and LM19 strains showed anti-inflammatory properties in TNF-α-stimulated HT-29 cells. Subsequently, bacterial strains exhibiting antioxidant and anti-inflammatory properties were tested in vivo in a mouse model with dinitrobenzene sulfonic acid (DNBS)-induced chronic colitis. Weight loss, intestinal permeability, and cytokine profiles were measured in mice as indicators of inflammation. One of the selected strains, Lactobacillus plantarum LM17, improved the health of the mice, as observed by reduced weight loss, and significantly decreased intestinal permeability. Altogether, our results demonstrate the potential of LAB (and lactobacilli in particular) isolated from the agave fermentation stage in mezcal production. Lactobacillus rhamnosus LM07 and Lactobacillus plantarum LM17 strains represent potential candidates for developing new probiotic supplements to treat inflammatory bowel disease (IBD).

Butyrate mediates anti-inflammatory effects of Faecalibacterium prausnitzii in intestinal epithelial cells through Dact3.

The commensal bacterium Faecalibacterium prausnitzii plays a key role in inflammatory bowel disease (IBD) pathogenesis and serves as a general health biomarker in humans. However, the host molecular mechanisms that underlie its anti-inflammatory effects remain unknown. In this study we performed a transcriptomic approach on human intestinal epithelial cells (HT-29) stimulated with TNF-α and exposed to F. prausnitzii culture supernatant (SN) in order to determine the impact of this commensal bacterium on intestinal epithelial cells. Moreover, modulation of the most upregulated gene after F. prausnitzii SN contact was validated both in vitro and in vivo. Our results showed that F. prausnitzii SN upregulates the expression of Dact3, a gene linked to the Wnt/JNK pathway. Interestingly, when we silenced Dact3 expression, the effect of F. prausnitzii SN was lost. Butyrate was identified as the F. prausnitzii effector responsible for Dact3 modulation. Dact3 upregulation was also validated in vivo in both healthy and inflamed mice treated with either F. prausnitzii SN or the live bacteria, respectively. Finally, we demonstrated by colon transcriptomics that gut microbiota directly influences Dact3 expression. This study provides new clues about the host molecular mechanisms involved in the anti-inflammatory effects of the beneficial commensal bacterium F. prausnitzii.

Gut Microbiota Abrogates Anti-α-Gal IgA Response in Lungs and Protects against Experimental Aspergillus Infection in Poultry.

Naturally occurring human antibodies (Abs) of the isotypes IgM and IgG and reactive to the galactose-α-1,3-galactose (α-Gal) epitope are associated with protection against infectious diseases, caused by pathogens expressing the glycan. Gut microbiota bacteria expressing α-Gal regulate the immune response to this glycan in animals lacking endogenous α-Gal. Here, we asked whether the production of anti-α-Gal Abs in response to microbiota stimulation in birds, confers protection against infection by Aspergillus fumigatus, a major fungal pathogen that expresses α-Gal in its surface. We demonstrated that the oral administration of Escherichia coli O86:B7 strain, a bacterium with high α-Gal content, reduces the occurrence of granulomas in lungs and protects turkeys from developing acute aspergillosis. Surprisingly, the protective effect of E. coli O86:B7 was not associated with an increase in circulating anti-α-Gal IgY levels, but with a striking reduction of anti-α-Gal IgA in the lungs of infected turkeys. Subcutaneous immunization against α-Gal did not induce a significant reduction of lung anti-α-Gal IgA and failed to protect against an infectious challenge with A. fumigatus. Oral administration of E. coli O86:B7 was not associated with the upregulation of lung cytokines upon A. fumigatus infection. We concluded that the oral administration of bacteria expressing high levels of α-Gal decreases the levels of lung anti-α-Gal IgA, which are mediators of inflammation and lung damage during acute aspergillosis.

Anti-tumoral Effects of Recombinant Lactococcus lactis Strain Secreting IL-17A Cytokine.

Interleukin-17A (IL-17A) is a pro-inflammatory cytokine produced by TH17 cells that participates and contributes in host defense and autoimmune disease. We have recently reported antitumor properties of the probiotic strain of Lactobacillus casei BL23 in mice and TH17 cells was shown to play an important role in this beneficial effect. In order to better understand the role of IL-17A in cancer, we constructed a recombinant strain of Lactococcus lactis producing this cytokine and we determined its biological activity in: (i) a bioassay test for the induction of IL-6 production by murine fibroblasts 3T3 L1 cells line and (ii) in a mouse allograft model of human papilloma virus (HPV)-induced cancer. Our data show that recombinant L. lactis produces and efficiently secretes biologically active IL-17A cytokine. Interestingly, ∼26% of mice intranasally treated with L. lactis-IL-17A and challenged with TC-1 cells remained tumor free over the experiment, in contrast to control mice treated with the wild type strain of L. lactis which developed 100% of aggressive tumors. In addition, the median size of the ∼74% tumor-bearing mice treated with recombinant L. lactis-IL-17A, was significantly lower than mice treated with L. lactis-wt. Altogether, our results demonstrate that intranasal administration with L. lactis secreting IL-17A results in a partial protection against TC-1-induced tumors in mice, confirming antitumor effects of this cytokine in our cancer model.

Elucidating the Immune-Related Mechanisms by Which Probiotic Strain Lactobacillus casei BL23 Displays Anti-tumoral Properties.

We have recently described antitumor properties of Lactobacillus casei BL23 strain in both a mouse allograft model of human papilloma virus (HPV)-induced cancer and dimethylhydrazine-associated colorectal cancer. However, the mechanisms underlying these beneficial effects are still unknown. Interestingly, in vitro cellular models show that this bacterium is able to stimulate the production of high levels of IL-2. Because this cytokine has well-known antitumor properties, we decided to explore its role in the anti-cancer effects of BL23 using the HPV-induced cancer model. We found a negative correlation between IL-2 and tumor size confirming the necessity of IL-2 to protect from tumor development. Then, we blocked IL-2 synthesis using neutralizing monoclonal antibodies in mice that were challenged with lethal levels of tumor cells; this led to a significant reduction in the protective abilities of BL23. Next, we used a genetically modified strain of Lactococcus lactis to deliver exogenous IL-2 to the system, and in doing so, we were able to partially mimic the antitumor properties of BL23. Additionally, we showed the systemic role of T-cells in tumor protection through a negative correlation between tumor size and T-cells subpopulations and an increasement of BL23-specific local Foxp3 levels in tumor-bearing mice. Finally, we observed a negative correlation between tumor size and NK+ cells, but local recruitment of NK cells and cytotoxic activity appeared specific to BL23 treatment. Taken together, our data suggest that IL-2 signaling pathway plays an important role in the anti-tumoral effects of probiotic strain L. casei BL23. These results encourage further investigation in the use of probiotic strains for potential therapeutic applications to clinical practice, in particular for the treatment of colorectal cancer. Furthermore, our approach could be extended and applied to other potential beneficial microorganisms, such as gut microbiota, in order to better understand the crosstalk between microbes and the host.

Identification of novel anti-inflammatory probiotic strains isolated from pulque.

Probiotics are live microorganisms which when administered in adequate amounts, confer health benefits on the host. Their use is more and more widespread for both prevention and treatment of diseases, including traveler's diarrhea and inflammatory bowel diseases (IBDs). In this work, we isolated and characterized novel candidate probiotic strains from pulque (xaxtle), a traditional Mexican alcoholic fermented beverage. A total of 14 strains were obtained from xaxtle samples isolated from three different Mexican regions. Species identification was performed by biochemical methods and 16S rRNA gene targeted PCR. The isolates belonged to the Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus brevis, and Lactobacillus composti phylogenetic groups, with L. brevis being the most dominant group. Bacteria were tested for lysozyme, low pH, and bile acid resistance. Moreover, the strains were tested for adherence to human intestinal epithelial cells and screened for their immunomodulatory properties using a cellular model. Selected bacterial strains with anti-inflammatory properties were then tested in vivo in a dinitro-benzene sulfonic acid (DNBS)-induced chronic colitis mouse model, and weight loss, gut permeability, and cytokine profiles were measured as readouts of inflammation. One of the selected strains, Lactobacillus sanfranciscensis LBH1068, improved mice health as observed by a reduction of weight loss, significant decreases in gut permeability, and cytokine modulation. Altogether, our results highlighted the potential of lactobacilli isolated from pulque and in particular the strain L. sanfranciscensis LBH1068 as a novel probiotic to treat IBD.