Project description:Emerging evidence supports that the efficacy of immune checkpoint blockade (ICB) therapy is associated with the host's gut microbiota, as prior antibiotic intake often leads to poor outcome and low responsiveness toward ICB treatment. Therefore, we hypothesized that the efficacy of ICB therapy like anti-programmed cell death protein-1 (PD-1) treatment required an intact host gut microbiota, and it was established that probiotics could enhance the recovery of gut microbiota disruption by external stimuli. Thus, the present study aimed to evaluate the effect of the probiotics, Lactobacillus rhamnosus Probio-M9, on recovering antibiotic-disrupted gut microbiota and its impact on the outcome of ICB therapy in tumor-bearing mice. We first disrupted the mouse microbiota by antibiotics and then remediated the gut microbiota by probiotics or naturally. Tumor transplantation was then performed, followed by anti-PD-1-based antitumor therapy. Changes in the fecal metagenomes and the tumor suppression effect were monitored during different stages of the experiment. Our results showed that Probio-M9 synergized with ICB therapy, significantly improving tumor inhibition compared with groups not receiving the probiotic treatment (P < 0.05 at most time points). The synergistic effect was accompanied by effective restoration of antibiotic-disrupted fecal microbiome that was characterized by a drastically reduced Shannon diversity value and shifted composition of dominating taxa. Moreover, probiotic administration significantly increased the relative abundance of beneficial bacteria (e.g., Bifidobacterium pseudolongum, Parabacteroides distasonis, and some Bacteroides species; 0.0001 < P < 0.05). The gut microbiome changes were accompanied by mild reshaping of the functional metagenomes characterized by enrichment in sugar degradation and vitamin and amino acid synthesis pathways. Collectively, this study supported that probiotic administration could enhance the efficacy and responsiveness of anti-PD-1-based immunotherapy, and Probio-M9 could be a potential candidate of microbe-based synergistic tumor therapeutics. The preclinical data obtained here would support the design of future human clinical trials for further consolidating the current findings and for safety assessment of probiotic adjunctive treatment in ICB therapy.

Project description:The study of microorganisms in outer space has focused mainly on investigating phenotypic changes in microbial pathogens induced by factors encountered in space. This study aimed to investigate the effect of space exposure on a probiotic bacterium, Lacticaseibacillus rhamnosus Probio-M9. Probio-M9 cells were exposed to space in a spaceflight. Interestingly, our results showed that a substantial proportion of space-exposed mutants (35/100) exhibited a ropy phenotype, characterized by their larger colony sizes and an acquired ability to produce capsular polysaccharide (CPS), compared with the original Probio-M9 or the ground control isolates without space exposure. Whole-genome sequencing analyses on both the Illumina and PacBio platforms revealed a skewed distribution of single nucleotide polymorphisms (12/89 [13.5%]) toward the CPS gene cluster, particularly in the wze (ywqD) gene. The wze gene encodes a putative tyrosine-protein kinase that regulates CPS expression through substrate phosphorylation. Transcriptomics analysis of two space-exposed ropy mutants revealed increased expression in the wze gene relative to a ground control isolate. Finally, we showed that the acquired ropy phenotype (CPS-producing ability) and space-induced genomic changes could be stably inherited. Our findings confirmed that the wze gene directly influences the capacity for CPS production in Probio-M9, and space mutagenesis is a potential strategy for inducing stable physiological changes in probiotics. IMPORTANCE This work investigated the effect of space exposure on a probiotic bacterium, Lacticaseibacillus rhamnosus Probio-M9. Interestingly, the space-exposed bacteria became capable of producing capsular polysaccharide (CPS). Some probiotic-derived CPSs have nutraceutical potential and bioactive properties. They also enhance the survival of probiotics through the gastrointestinal transit and ultimately strengthen the probiotic effects. Space mutagenesis seems to be a promising strategy for inducing stable changes in probiotics, and the obtained high-CPS-yielding mutants are valuable resources for future applications.

Project description:Water buffalo mastitis represents a major issue in terms of animal health, cost of therapy, premature culling and decreased milk yeld. The emergence of antibiotic resistance has led to investigate strategies to avoid or reduce antibiotics' based therapies, in particular during subclinical mastitis. The use of Generally Regarded As Safe bacteria (GRAS) such as Lactobacillus rhamnosus to restore the unbalance in mammary gland microbiota could provide potential corrective measures. The aim of this study was to investigate the changes in milk microbiota after the intramammary treatment with inactivated cultures of Lactobacillus rhamnosus of mammary gland quarters naturally affected by subclinical mastitis as compared to antibiotic therapy.A number of 43 quarters affected by subclinical mastitis with no signs of clinical inflammation and aerobic culture positive for pathogens were included in the study. The experimental design was as follows: 11 quarters were treated with antibiotics, 15 with inactivated cultures of Lactobacillus rhmnosus and 17 with PBS as negative control, by means of intrammary injection. Samples were collected at eight time points, pre- (T-29, T-21, T-15, T-7, T0 days) and post- treatment (T1, T2, and T6 days). Microbiological culture and Somatic Cell Count (SCC) were perfomed on all the samples, and microbiota was determined on milk samples collected at T0 and T6 by amplifying the V4 region of 16S rRNA gene by PCR and sequencing using next generation sequencing technique. Treatment with Lactobacillus rhamnosus elicited a strong chemotactic response, as determined by a significant increase of leukocytes in milk, but did not change the microbiological culture results of the treated quarters. For what concerns the analysis of the microbiota, the treatment with Lactobacillus rhamnosus induced the modification in relative abundance of some genera such as Pseudomonas and 5-7N15. As expected, antibiotic treatment caused major changes in microbiota structure with an increase of Methylobacterium relative abundance. No changes were detected after PBS treatment. In conclusion, the present findings demonstrated that the in vivo intrammmary treatment with Lactobacillus rhamnosus has a transient pro-inflammatory activity by increasing SCC and is capable to modify the microbiota of milk after six days from inoculation, albeit slightly, even when the bacterial cultures were heat inactivated. Further studies are necessary to assess the potential use of this GRAS as supportive therapy against mastitis.

Project description:A variety of foods fermented with lactic acid bacteria serve as dietary staples in many African communities; yet, their bacterial profiles are poorly characterized. The integration of health-promoting probiotics into naturally fermented milk products could make a profound impact on human health. Here, we characterize the bacterial community composition of a naturally fermented milk product (lait caillé) from northern Senegal, prepared in wooden bowls (lahals) with a bacterial biofilm to steer the fermentation process. We incorporated a probiotic starter culture containing the most documented probiotic strain Lactobacillus rhamnosus GG (generic strain name yoba 2012) into the local fermentation process. Bar-coded 16S rRNA amplicon sequencing of lait caillé samples indicated that the bacterial community of lait caillé has high species richness with over 100 bacterial genera; however, few have high abundance. In contrast to the diverse bacterial compositions of other characterized naturally fermented milk products, the composition of lait caillé predominantly consists of the lactic acid bacteria Streptococcus and Lactobacillus, resembling the bacterial composition in regular yogurt. The bacterial community composition of lait caillé varies geographically based on the presence of some genera, including Lactoccoccus, Enterococcus, Bifidobacterium, and Bacillus, but this trend is not consistent within production communities. The diversity of bacterial communities is much higher in the lahal biofilm than in the naturally fermented milk products, which is in turn greater than in commercial yogurts. Addition of a starter culture with L. rhamnosus yoba 2012 to milk in lahals led to substantial growth of this probiotic bacterium during the fermentation process. Two independent quantitative PCR-analyses specific for L. rhamnosus yoba 2012 indicated a 20- to 60-fold increase in the total number of probiotic bacteria in the first batch after inoculation. A similar increase of the probiotic was observed in a variation of lait caillé prepared with carbohydrate-rich millet granules (thiakry) added prior to fermentation. This study shows the feasibility of integrating health-promoting probiotic strains into naturally fermented foods produced in regions with a high prevalence of malnutrition.

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Inhibitory Effects of Breast Milk-Derived Lactobacillus rhamnosus Probio-M9 on Colitis-Associated Carcinogenesis by Restoration of the Gut Microbiota in a Mouse Model.

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