Prolonged antigen exposure in chronic viral infections reduces the effector capacity of cytotoxic T cells-a phenomenon known as T cell exhaustion. Development of T cell exhaustion is driven by high viral titers, strong TCR stimulation, and high antigen concentrations associated with strong inflammatory signals. A largely unexplored factor has been the influence of the microbiome in these processes. Here, we report that T cell exhaustion progresses independently of the presence or absence of a microbiome in chronic lymphocytic choriomeningitis virus (LCMV) infections. Virus-specific CD8 T cells in germ-free mice showed high expression of the inhibitory receptor PD-1 and decreased cytokine production. Moreover, their global gene expression patterns, as determined by single-cell sequencing, were similar to those of cells in specific pathogen-free mice. In line with this, we observed similar pathogen loads with and without a microbiome. Thus, our study demonstrates that the microbiome is dispensable for the induction of T cell exhaustion and for the limited virus control seen in chronic LCMV infections.

Background/Objectives: Ulcerative colitis (UC) is characterized by the interplay between immune responses and dysbiosis in disease development. Aiming to provide additional insights into disease development and potential treatment strategies, the present study investigates the local effect of oral treatment with polysaccharides obtained from Auricularia auricula’s submerged culture in an experimental model of DSS-induced colitis and its impact on lesion resolution. Methods: The structure and monosaccharide composition of Auricularia polysaccharides were characterized through Nuclear Magnetic Resonance (NMR). To evaluate the effect of this polysaccharide on the murine model, wild-type and Dectin-1 knockout mice were treated or not with the exopolysaccharide (EPS) while under DSS consumption. During the experimental period, feces samples were collected to evaluate microbial shifts during disease development, and, finally, the colonic tissue was analyzed to assess the inflammatory process and cytokine production. Results: The EPS composition showed a polymeric mixture of glucans and fucogalactomannans. The treatment of the wild-type DSS-induced colitis group improved the inflammatory response by increasing gut–homeostatic cytokines, such as interleukin-10 (IL-10) and tumor necrosis factor-alpha (TNF-α). The Dectin-1 KO mice group did not show the same enhancement after EPS treatment. The microbiome analysis revealed a difference in the genotype, and the treatment modified the DSS microbiome modulation, with nine and four ASVs in WT and Dectin-1 KO mice, respectively. Conclusions: The EPS treatment demonstrated therapeutic potential in treating inflammatory intestinal diseases by modulating cytokine secretion and microbiota composition, which is dependent on the Dectin-1 receptor’s carbohydrate recognition.

Background:Inflammatory Bowel Diseases (IBDs) are characterized by intestinal dysbiosis and immune dysregulation. Annexin A1 (AnxA1) promotes epithelial repair and inhibits immune responses during IBD. However, AnxA1's impact on gut microbiota during IBD remains unclear. Here, we experimentally investigated the microbiota profile during colitis in wild-type (WT) and AnxA1-deficient mice (AnxA1-/-), and evaluated an observational cohort in IBD patients with high or low AnxA1 expression. Methods:Colitis was induced in C57BL/6 WT and AnxA1 -/- mice via oral administration of 2% DSS for six days. Fecal samples were collected at baseline, peak inflammation (day 6), and during the recovery phase (day 10) for 16S rRNA sequencing. Human microbiota data from the Lifelines Dutch Microbiome Project cohort, including IBD and healthy subjects, were analyzed for AnxA1 expression using R software. Results:Healthy AnxA1-/- mice exhibited reduced microbial richness and a distinct gut microbiota composition, marked by increased Proteobacteria and Parasutterella, and reduced Deferribacterota, Campylobacterota, and Verrucomicrobiota. During DSS-induced colitis, AnxA1-/- mice showed greater weight loss and heightened inflammation, displaying earlier and more pronounced microbial shifts, including increased Proteobacteria, Cyanobacteria, Parabacteroides, Bacteroides, and Escherichia-Shigella. In contrast, WT mice exhibited delayed changes, with expansion of Alloprevotella, Akkermansia, and Faecalibaculum after day 6. In human IBD samples, Crohn's disease (CD) patients with low AnxA1 expression and active inflammation presented an altered microbiota enriched in Lachnoclostridium and Parabacteroides, while ulcerative colitis (UC) patients showed phylum-level shifts modulated by AnxA1 levels. Notably, non-inflamed CD and UC patients with low AnxA1 differed significantly in microbiota composition. Moreover, inflamed CD patients with high AnxA1 expression showed microbial profiles resembling those of healthy controls, while low AnxA1 expression was associated with a more pronounced dysbiotic state. Conclusion:AnxA1 is implicated in microbiota control under healthy and IBD conditions. Accordingly, the microbiota of healthy AnxA1-/- mice, colitic AnxA1-/- mice, and IBD patients with low AnxA1 expression exhibit dysbiosis compared to their respective controls. Together, these unprecedented findings reveal AnxA1 as a potential regulatory protein in the immune-microbiota axis involved in IBD pathogenesis.

Minas Gerais is known for producing raw milk artisanal cheeses, particularly Serra da Canastra. These cheeses are traditionally produced using a natural whey starter culture (NWS) known as pingo. The role of pingo in shaping cheese characteristics remains poorly understood. This research evaluated the microbiological and physicochemical effects of pingo on the artisanal cheese using a laboratory-scale model. Experimental cheeses were produced with pingo using two input frameworks: raw milk with high bacterial content and thermized milk with lower bacterial content. Control cheeses were made without pingo. Samples were analyzed during production and over 60 days of ripening for microbial counts (lactic acid bacteria (LAB), coliforms and Escherichia coli), pH, aw, acidity and NaCl content. The microbial composition was determined by 16S rRNA sequencing. The logistic model was adjusted during 24 h of manufacture and the Weibull model was adjusted during ripening. Results indicated that during the production of the cheeses the maximum growth rate (μmax) for E. coli was 25 % lower in cheeses produced with thermized milk and NWS compared to cheese without the addition of NWS. In raw milk cheeses, μmax for E. coli was 2.3 to 3.0 times higher and no effect of NWS was observed, suggesting that NWS inhibits the growth of indicator microorganisms during cheese manufacturing only when the initial contaminant count is low. The time for the first decimal reduction (δ) of E. coli and coliforms during cheese ripening decreased by half with the addition of pingo, regardless of the type of milk used. The main genera identified in NWS was Streptococcus and a low relative abundance of Lactococcus. Cheeses produced with NWS presented a high prevalence of Streptococcus, followed by Lactococcus and Lactobacillus. In contrast, the cheese produced with raw milk and without NWS presented a high prevalence of Lactococcus. This study highlights the crucial role of pingo in reducing microbial loads and enhancing cheese safety, particularly when high- quality milk with low bacterial content is used, but its effectiveness is limited in poor-quality milk frameworks.

Objectives: To evaluate the associations between the quality of the diet and its components and microbial diversity and composition in peritoneal dialysis (PD) patients. Design and Methods: This cross-sectional study included PD patients for at least 3 months, aged 18-75 years and clinically stable. The Diet Quality Index (DQI), validated for the Brazilian population, is based on the energy density of 11 components ("sugar and sweets;""beef, pork, and processed meat;""refined grains and breads;""animal fat;""poultry, fish, and eggs;""whole cereals, tubers, and roots;""fruits"; "nonstarch vegetables;""legumes and nuts;""milk and dairy products;"and "vegetable oil"). A proportional score-based on the adequacy of the intake to Brazilian dietary guidelines-is calculated, and a final score ranged from 0 (worse) to 100 (better) is obtained. Fecal samples were collected at home, in a sterile material, kept refrigerated, and delivered to the clinic within 12 hours; a-diversity indices (Observed operational taxonomic units, Chao-1, Shannon's, Gini-Simpson's, Pielou eveness, and Faith phylogenetics) and microbial profile were determined by 16S ribosomal DNA with polymerase chain reaction-amplification and sequenced on an Illumina MiSeq platform. Results: Forty-three participants were included (53.5% men, 52.4 +/- 14.1 years, body mass index: 25.9 +/- 4.1 kg/m2, 30.2% had diabetes mellitus). DQI score was 50.5 (41.9-54.9). The lowest energy density was for the components "animal fat"and "whole cereals and breads, tubers, and roots,"and the highest were for "refined grains and bread"and "beef, pork, and processed meat."Diversity indices and Enterorhabdus genus were directly associated with the energy density of the components "whole cereals and breads, tubers, and roots"and inversely with "refined grains and bread,"after adjustments for age and diabetes. Conclusions: Even low, the intake of whole cereals, tubers, and roots has the potential to positively influence the microbiota profile in PD patients.

Microbial ecosystems are fundamental to planetary and human health, yet human activities are accelerating their loss. Disruptions to microbial communities undermine environmental stability, biodiversity, and health. Urgent action is required to preserve microbial diversity. The Microbiota Vault Initiative provides a global framework to safeguard microbiomes from human, animal, and environmental sources. It proactively archives microbial diversity for future needs, prioritizing depositor sovereignty, equitable collaboration, and ethical governance. By sharing limited information on deposits, the initiative fosters microbial conservation and collaboration between local and global researchers. It complements other efforts to ensure the resilience of microbiomes in an era of rapid environmental change.

The intestinal microbiota is shaped by fiber-rich ingredients, such as unripe banana flour (UBF), high in resistant starch (RS). We investigated the effects of RS-rich UBF and inulin on gut microbiota and intestinal function in a double-blind, randomized, placebo-controlled pilot trial. Forty-eight healthy adults consumed maltodextrin (control), inulin, or UBF three times weekly for six weeks. Microbiota composition and function were analyzed using 16S rRNA gene sequencing and PICRUSt, alongside fecal short-chain fatty acids, blood biochemistry, and gastrointestinal parameters. We observed two microbiota clusters at baseline, one Prevotella-rich (P) and one Bacteroides-rich (B), with distinct responses to the interventions. Only cluster P subjects consuming UBF showed significant global microbiota shifts (weighted Unifrac Beta diversity, PERMANOVA p = 0.007) and major functional changes (533 KEGG orthologs, FDR < 0.05). Inulin produced modest modulation (19 KOs) on cluster P, and no effects were observed on cluster B. RS-rich UBF modulated gut microbiota in a composition-dependent manner, supporting the potential of microbiota-based stratification to improve dietary fiber interventions.

Magnesium (Mg2+) is essential for life, and low levels impair immune function, promote chronic inflammation, and influence the intestinal microbiome, with the peritoneal cavity serving as a site for direct interaction between the cavity and intestinal contents, including the microbiota. This study investigates the effects of a Mg2+-restricted diet on peritoneal immune cells and its interplay with the intestinal microbiome. Male C57BL/6NTaq mice were divided into three groups: control, restricted, and restored. The control group received a diet containing 500 mg Mg2+/kg, the restricted group received a diet with 50 mg Mg2+/kg for four weeks, and the restored group first received the restricted diet for four weeks, followed by the control diet supplemented with 0.5 g MgCl₂ per liter of water for an additional four weeks. Results showed Mg2+ restriction did not affect body weight, food intake, or water consumption but induced hypomagnesemia, reversible upon dietary restoration. Mg2+ deficiency increased in neutrophils numbers in the blood and peritoneal cavity, indicating an inflammatory response. Gene expression analysis in peritoneal mononuclear cells revealed elevated levels of Nfkb, Stat1 and Stat3, suggesting heightened inflammatory signaling. Additionally, cytokine expression analysis showed increased levels of Tnfa, Il1b and Il10, but not Il6, in Mg2+-restricted group. The intestinal microbiome of Mg2+-restricted mice exhibited increased alpha diversity, with changes in taxa abundance, including an increase in Romboutsia ilealis and a decrease in the Oscillospiraceae and Lachnospiraceae. Mg2+ deficiency significantly affects some immune functions and gut microbiota, highlighting the importance of Mg²+ in maintaining the gut health.

The aim of this study was to investigate the vertical transfer of microbiota from dams to the offspring. We studied a pair of 20 dams and its offspring. Maternal sources (colostrum, feces and vaginal secretion) and newborn fecal samples were analyzed using 16S rDNA amplicon sequencing on days 1, 3, 7, 14 and 28. Overall, newborns were maintained healthy and did not receive antimicrobial therapy. The Source Tracker analysis indicated that the newborn fecal microbiota was similar to colostrum and vaginal secretion from day 1 up to 7. However, an unknown source (probably from the environment) showed a gradual increase in its similarity with fecal samples from calves measured from day 3 to 28. The most abundant bacteria groups on meconium (day 1) and calf fecal samples on day 3 were Escherichia-Shigella and Clostridium, respectively. On day 7, the predominant genus were Bifidobacterium and Lactobacillus, while Fusobacterium was the most abundant genus on day 14, coinciding with the diarrhea peak. Faecalibacterium showed a gradual increase throughout the neonatal period. Maternal sources contribute to the neonatal microbiota, however other unknown sources (probably environment) had a strong influence on development of the gut microbiota later in the neonate period.

Macrophages play a key role in disseminated cryptococcosis, a deadly fungal disease caused by Cryptococcus neoformans. This opportunistic infection can arise following the reactivation of a poorly characterized latent infection attributed to dormant C. neoformans. Here, we investigated the mechanisms underlying reactivation of dormant C. neoformans using an in vitro co-culture model of viable but non-culturable (VBNC; equivalent of dormant) yeast cells with bone marrow-derived murine macrophages (BMDMs). Comparative transcriptome analysis of BMDMs incubated with log, stationary phase or VBNC cells of C. neoformans showed that VBNC cells elicited a reduced transcriptional modification of the macrophage but retaining the ability to regulate genes important for immune response, such as NLRP3 inflammasome-related genes. We further confirmed the maintenance of the low immunostimulatory capacity of VBNC cells using multiplex cytokine profiling, and analysis of cell wall composition and dectin-1 ligands exposure. In addition, we evaluated the effects of classic (M1) or alternative (M2) macrophage polarization on VBNC cells. We observed that intracellular residence sustained dormancy, regardless of the polarization state of macrophages and despite indirect detection of pantothenic acid (or its derivatives), a known reactivator for VBNC cells, in the C. neoformans-containing phagolysosome. Notably, M0 and M2, but not M1 macrophages, induced extracellular reactivation of VBNC cells by the secretion of extracellular vesicles and non-lytic exocytosis. Our results indicate that VBNC cells retain the low immunostimulatory profile required for persistence of C. neoformans in the host. We also describe a pro-pathogen role of macrophage-derived extracellular vesicles in C. neoformans infection and reinforce the impact of non-lytic exocytosis and the macrophage profile on the pathophysiology of cryptococcosis.

Magnetic resonance imaging (MRI) is widely used for ischemic stroke lesion detection in mice. A challenge is that lesion segmentation often relies on manual tracing by trained experts, which is labor-intensive, time-consuming, and prone to inter- and intra-rater variability. Here, we present a fully automated ischemic stroke lesion segmentation method for mouse T2-weighted MRI data. As an end-to-end deep learning approach, the automated lesion segmentation requires very little preprocessing and works directly on the raw MRI scans. We randomly split a large dataset of 382 MRI scans into a subset (n = 293) to train the automated lesion segmentation and a subset (n = 89) to evaluate its performance. We compared Dice coefficients and accuracy of lesion volume against manual segmentation, as well as its performance on an independent dataset from an open repository with different imaging characteristics. The automated lesion segmentation produced segmentation masks with a smooth, compact, and realistic appearance that are in high agreement with manual segmentation. We report dice scores higher than the agreement between two human raters reported in previous studies, highlighting the ability to remove individual human bias and standardize the process across research studies and centers.

Blood orange juice is an important source of flavanones and anthocyanins, mainly hesperidin, narirutin, and cyanidin-3-O-glucoside. The benefits of these bioactive compounds have been reported, but the mechanistic details behind their biological effects are not well established. This study investigated the effects of Moro orange (Citrus sinensis L. Osbeck) juice (MOJ) on gut microbiota composition and cardiometabolic biomarkers in overweight women. In this study, 12 overweight women (BMI from 25.0 to 29.9 kg/m2), aged 18–37 years, consumed 500 mL of MOJ every day for 4 weeks. We assessed the gut microbiota composition, levels of short-chain fatty acids (SCFAs), cardiometabolic biomarkers, and insulin resistance (HOMA-IR) at baseline and after 2 weeks and 4 weeks of MOJ intake. The results suggested that MOJ intake affected the abundance of specific operational taxonomic units (OTUs) of the gut microbiota but did not significantly alter the diversity and general composition of the gut microbiota. However, MOJ intake increased the production of SCFAs, especially propionic and isobutyric acids, and significantly improved cardiometabolic biomarkers such as blood pressure and plasma VCAM-1 levels in the overweight women. Additionally, we observed significant associations between gut microbiota OTUs belonging to the Bacteroidetes phyla and Prevotella 9 genera and the cardiometabolic biomarkers. Furthermore, MOJ reduced fasting glucose and insulin levels and HOMA-IR values, thereby enhancing insulin sensitivity in the insulin-resistant overweight women. Finally, we highlighted the importance of orange juice intake duration because some beneficial changes such as blood pressure improvements were evident at the 2-week time interval of the intervention, but other changes became significant only at the 4-week interval of MOJ intake. In conclusion, our study demonstrated that changes in specific OTUs of the gut microbiota in response to MOJ intake were associated with significant improvements in some cardiometabolic biomarkers and SCFA levels in overweight women with insulin resistance.

Paracrine cerebral Interleukin 6 (Il6) is relevant for stroke recovery, but systemic Il6 elevation may worsen outcome. Hence, paracrine Il6 response modulation within the neurovascular unit has emerged as an attractive therapeutic approach. Lithium modulates Il6 responses and improves stroke outcome. However, lithium may cause serious adverse effects. Here, we report that Zincfinger protein 580 (Zfp580) mediates the effects of lithium on Il6 signaling. In contrast to lithium, Zfp580 inactivation had no neurotoxic effects, and Zfp580 knock out mice showed no phenotypic changes in cognitive and motor function behavioral tests. We discovered that lithium and hypoxia disinhibited Il6 via Zfp580 suppression and post-translational modification by small ubiquitin-like modifier (SUMO). After transient middle cerebral artery occlusion, loss of Zfp580 reduced paracrine Il6 and increased Il6 trans-signaling. Aside from modulating Il6 signaling, Zfp580 loss improved endothelial resilience to ischemia, was highly neuroprotective resulting in smaller infarcts and enhanced use-dependent neuroplasticity, all of which led to improved functional outcome. In conclusion, inactivation of Zfp580 exerts positive effects on multiple key mechanisms without exhibiting relevant adverse side effects, making it potentially a more specific and effective treatment target for stroke recovery than lithium. To fully assess its potential, Zfp580 inhibitors must be developed.

BACKGROUND: Prediction of poststroke outcome using the degree of subacute deficit or magnetic resonance imaging is well studied in humans. While mice are the most commonly used animals in preclinical stroke research, systematic analysis of outcome predictors is lacking. METHODS: We intended to incorporate heterogeneity into our retrospective study to broaden the applicability of our findings and prediction tools. We therefore analyzed the effect of 30, 45, and 60 minutes of arterial occlusion on the variance of stroke volumes. Next, we built a heterogeneous cohort of 215 mice using data from 15 studies that included 45 minutes of middle cerebral artery occlusion and various genotypes. Motor function was measured using a modified protocol for the staircase test of skilled reaching. Phases of subacute and residual deficit were defined. Magnetic resonance images of stroke lesions were coregistered on the Allen Mouse Brain Atlas to characterize stroke topology. Different random forest prediction models that either used motor-functional deficit or imaging parameters were generated for the subacute and residual deficits. RESULTS: Variance of stroke volumes was increased by 45 minutes of arterial occlusion compared with 60 minutes. The inclusion of various genotypes enhanced heterogeneity further. We detected both a subacute and residual motor-functional deficit after stroke in mice and different recovery trajectories could be observed. In mice with small cortical lesions, lesion volume was the best predictor of the subacute deficit. The residual deficit could be predicted most accurately by the degree of the subacute deficit. When using imaging parameters for the prediction of the residual deficit, including information about the lesion topology increased prediction accuracy. A subset of anatomic regions within the ischemic lesion had particular impact on the prediction of long-term outcomes. Prediction accuracy depended on the degree of functional impairment. CONCLUSIONS: For the first time, we developed and validated a robust tool for the prediction of functional outcomes after experimental stroke in mice using a large and genetically heterogeneous cohort. These results are discussed in light of study design and imaging limitations. In the future, using outcome prediction can improve the design of preclinical studies and guide intervention decisions.

Background Differences in patients gut microbiota composition with the potential for dysbiosis have been associated with chronic kidney disease (CKD). However, factors other than the disease itself, such as diet and cohabitation, have not been evaluated when gut microbiota of CKD patients was compared with that of healthy controls. The aim of this study was to compare the gut microbiota composition between patients on peritoneal dialysis (PD) and age-matched household contacts with normal renal function. Methods Fecal samples were collected from 20 patients [men: 70%; age: 53.5 years (48.2–66; median and interquartile range); length on PD: 14 months (5.2–43.5) and 20 controls. The region V4 of the 16S ribosomal RNA gene was PCR-amplified and sequenced on Illumina MiSeq platform. Dietary intake and diet quality were assessed by a 3-day food record and a diet quality index, respectively. Results No difference was found between the gut microbiota composition of patients and controls, assessed by alpha and beta diversities ( p > 0.05) and genera differential abundance ( p > 0.05). The most abundant phyla in both groups were Firmicutes (PD = 45%; Control: 47%; p = 0.65) and Bacteroidetes (PD = 41%; Control: 45%; p = 0.17). The phylum Proteobacteria, known as a potential marker of gut dysbiosis, was not different in proportions between groups ( p > 0.05). No difference was observed regarding diet quality and dietary intake of fiber, protein and other nutrients ( p > 0.05). Conclusion Gut microbiota of patients on PD did not differ from household contacts. This result suggests that cohabitation and dietary intake might have outweighed the disease influence on gut microbiota composition of our PD patients.

Insulin-like growth factor 1 (Igf1) and insulin-like growth factor binding protein 3 (Igfbp3) are endocrine and paracrine factors that influence stroke occurrence, severity, and recovery. Low levels of endocrine Igf1 and Igfbp3 were associated with larger infarct volumes and unfavorable outcomes. Paracrine Igf1 is brain cytoprotective and improves functional recovery after stroke. In this study, we evaluated the effects of zinc finger protein 580 (Zfp580) on endocrine and paracrine Igf1 and Igfbp3 after stroke. Zfp580 suppressed the expression of Igf1 and Igfbp3 in cerebral microvascular endothelial cells (bEnd.3) as determined by real-time RT-PCR. Zfp580 was suppressed by combined oxygen and glucose deprivation (OGD) and mediated the effect of OGD on Igf1 and Igfbp3. In vivo, we evaluated paracrine regulation by real-time RT-PCR of brain lysates and endocrine regulation by ELISA of blood samples. Genomic ablation of Zfp580 did not alter basal paracrine or endocrine Igf1 and Igfbp3 levels. After transient middle cerebral artery occlusion (MCAo), Zfp580 was globally elevated in the brain for up to 3 days. Paracrine Igf1 and Igfbp3 were selectively induced in the ischemic hemisphere from day 2 to day 3 or day 1 to day 7, respectively. In Zfp580 knockout mice, the paracrine regulations of Igf1 and Igfbp3 were attenuated while endocrine Igf1 and the molar Igf1/Igfbp3 ratio were increased. In conclusion, Zfp580 differentially controls paracrine and endocrine Igf1 and Igfbp3 after stroke. Inhibition of Zfp580 might be a new treatment target leading to increased activity of Igf1 to improve stroke outcome.

The lung epithelial barrier serves as a guardian towards environmental insults and responds to allergen encounter with a cascade of immune reactions that can possibly lead to inflammation. Whether the environmental sensor aryl hydrocarbon receptor (AhR) together with its downstream targets cytochrome P450 (CYP1) family members contribute to the regulation of allergic airway inflammation remains unexplored. By employing knockout mice for AhR and for single CYP1 family members, we found that AhR -/- and CYP1B1 -/- but not CYP1A1 -/- or CYP1A2 -/- animals display enhanced allergic airway inflammation compared to WT. Expression analysis, immunofluorescence staining of murine and human lung sections and bone marrow chimeras suggest an important role of CYP1B1 in non-hematopoietic lung epithelial cells to prevent exacerbation of allergic airway inflammation. Transcriptional analysis of murine and human lung epithelial cells indicates a functional link of AhR to barrier protection/inflammatory mediator signaling upon allergen challenge. In contrast, CYP1B1 deficiency leads to enhanced expression and activity of CYP1A1 in lung epithelial cells and to an increased availability of the AhR ligand kynurenic acid following allergen challenge. Thus, differential CYP1 family member expression and signaling via the AhR in epithelial cells represents an immunoregulatory layer protecting the lung from exacerbation of allergic airway inflammation.

Our work demonstrated a dynamic yet stable microbial ecosystem during cheese production using an endogenous starter culture. This was observed across several distinct producers and was marked by genomic evidence of continued phage-bacterium interactions, such as the presence of bacterial defense mechanisms.

Boza is a traditional low-alcohol fermented beverage from the Balkan Peninsula, frequently explored as a functional food product. The product is rich in Lactic Acid Bacteria (LAB) and some of them can produce bacteriocins. In this study, a sample of Boza from Belogratchik, Bulgaria, was analyzed for the presence of bacteriocinogenic LAB, and after analyses by RAPD-PCR, three representative isolates were characterized by genomic analyses, using whole genome sequencing. Isolates identified as Pediococcus pentosaceus ST75BZ and Pediococcus pentosaceus ST87BZ contained operons encoding for bacteriocins pediocin PA-1 and penocin A, while isolate identified as Pediococcus acidilactici ST31BZ contained only the operon for pediocin PA-1 and a CRISPR/Cas system for protection against bacteriophage infection. The antimicrobial activity of bacteriocins produced by the three isolates was inhibited by treatment of the cell-free supernatants with proteolytic enzymes. The produced bacteriocins inhibited the growth of Listeria monocytogenes , Enterococcus spp. and some Lactobacillus spp., among other tested species. The levels of bacteriocin production varied from 3200 to 12,800 AU/ml recorded against L. monocytogenes 104, 637 and 711, measured at 24 h of incubation at 37 °C. All bacteriocins remained active after incubation at pH 2.0–10.0. The activity mode of the studied bacteriocins was bactericidal, as determined against L. monocytogenes 104, 637 and 711. In addition, bactericidal activity was demonstrated using a cell leakage β-galactosidase assay, indicating a pore formation mechanism as a mode of action. The present study highlights the importance of combining genomic analyses and traditional microbiological approaches as way of characterizing microbial interactions in fermented foods.