Exposure to cadmium (Cd), a toxic heavy metal, is a severe threat to organismal health, causing a wide range of pathological alterations in various tissues and organs. Alterations in the composition and function of the gut microbiome have been indicated across numerous animals exposed to Cd. However, the impact of Cd inhalation exposure on the pulmonary microbiome has not been well investigated yet. Therefore, in this study, we investigated the effects of exposure to CdONPs and its clearance on both colonic and pulmonary microbiomes in mice. The diversity of both colonic and pulmonary microbiomes of exposed mice was significantly affected after 9 weeks of CdONPs inhalation. The effects of CdONPs exposure on bacterial composition and function were more pronounced in the colonic microbiome than in the pulmonary microbiome. The clearance was more efficient in the restoration of gut microbiome composition in comparison to the lung microbiome. Moreover, we evaluated a bidirectional interaction between Cd exposure and gut microbiota. Duncaniella, Odoribacter, and Pontibacter were the prominent biomarkers that significantly positively correlated with dysregulated functions in the colonic microbiome of exposed mice. Based on the PICRUSt2 prediction analysis, our results suggested that perturbations in the gut microbiota balance due to Cd exposure were associated with the increase in the proportion level of bacteria with excessive membrane transporters, which may potentially augment the absorption of this metal by intestinal microbiota thereby leading to the accumulation of Cd in intestinal bacteria and the potential alleviation of the Cd toxicity effect. Furthermore, genes related to metal chelators were consistent with the colonic microbiome of exposed mice, suggesting possible promotion of Cd excretion and its eventual fecal elimination. However, these observations derived from 16S rRNA profiling and PICRUSt2 predictions would need to be verified experimentally to establish any functional or mechanistic implications. This could be considered a key factor in determining the intestinal bacterial species able to minimize the toxicity of heavy metals in future therapeutic approaches. • Inhalation of CdO nanoparticles significantly alters both gut and pulmonary microbiome composition and diversity in mice. • Microbiome changes are more pronounced in the gut than in the lungs following inhalation exposure. • Partial recovery of microbiome composition occurs after the clearance period, with greater restoration in the gut than in the lung. • Predicted functional profiles indicate shifts in microbial metabolic potential associated with Cd exposure. • The findings support a potential interaction between inhaled Cd exposure and the gut microbiome, highlighting the relevance of the gut–lung axis.

This study investigated the effects of dietary inclusion of corn and peas on the fecal microbiota composition and functional potential in Sarda goats. Twelve female goats were allocated to two groups, a group grazing on a natural pasture (GP) and a group grazing on the same pasture with a diet enriched with corn and peas (GC), both grain supplements at 100 g/day. Fecal samples were collected, freeze-dried and subjected to 16S rRNA gene sequencing of the V4-V5 region, followed by bioinformatic and statistical analyses. Alpha diversity metrics indicated a significant reduction in microbial richness and phylogenetic diversity in the GC group, and beta diversity analysis revealed distinct community clustering. Taxonomic profiling showed Firmicutes and Bacteroidota as the dominant phyla, with a significantly altered Firmicutes/Bacteroidota (F/B) ratio in the GC group. LEfSe analysis identified 31 differentially abundant taxa between the groups. Functional profiling revealed 28 KEGG Level 3 pathways enriched in both groups. Pathways related to cell growth and motility were more abundant in the GP group, while the GC was enriched in amino acid metabolism and membrane-associated processes. These findings suggest that dietary supplementation with plant-based concentrates modulates both the taxonomic structure and functional potential of the fecal microbiota in goats.

AIMS:The clinical effectiveness of antibiotic (ATB) therapy in patients with infected diabetic foot ulcers (iDFUs) depends on achieving sufficient ATB concentrations in both serum and peripheral tissues. This study evaluated the availability and bactericidal activity of the time-dependent ATBs-ceftazidime (CTZ) and amoxicillin/clavulanate (AMC) in serum and peripheral tissues by comparing bolus versus continuous administration. METHODS:Sixty patients with iDFUs were randomised into four subgroups according to ATB and administration method: CTZ bolus (CTZbolus), CTZ continuous infusion (CTZcontinuous), AMC bolus (AMCbolus), and AMC continuous infusion (AMCcontinuous). Once steady-state concentrations were reached, microdialysis was used to assess ATB levels in tissue adjacent to the ulcer. Serum and tissue samples were collected over a 6-h period. Bactericidal activity was assessed by peak concentrations (Cmax), area under the curve (AUC), and the proportion of time free drug concentrations remained above the minimum inhibitory concentration (fT > MIC). Target thresholds were fT > MIC ≥ 60% for CTZ and ≥ 50% for AMC. RESULTS:All patients achieved serum fT > MIC targets for CTZ and AMC in 100% of AMCbolus and 89% of AMCcontinuous. In tissue, target attainment was 60% (CTZbolus), 73% (CTZcontinuous), 79% (AMCbolus), and 83% (AMCcontinuous). Bolus administration led to rapid serum peaks (5 min) and delayed tissue peaks (30-60 min), with subsequent declines. Continuous infusion produced gradual concentration increases over time. CONCLUSIONS:While all patients achieved serum fT > MIC targets for CTZ and AMC (100% for AMCbolus and 89% for AMCcontinuous), target attainment in tissue was substantially lower. These findings suggest that although serum pharmacodynamic targets are reliably achieved, tissue exposure remains suboptimal.

This study aimed to compare the antimicrobial activity of different classes of antidepressants, specifically Brintellix (vortioxetine), a serotonin modulator and stimulator (SMS), and Cipralex (escitalopram) and Seropram (citalopram), both selective serotonin reuptake inhibitors (SSRIs). The effects of these antidepressants on growth kinetics were tested in 22 strains of the genus Bifidobacterium . Species were cultivated in TPY liquid medium under anaerobic conditions. Each antidepressant, in its original commercially available liquid form, was added to the culture medium at final concentrations of 200, 400, and 600 µg/mL. Bacterial growth was monitored via OD₆₀₀, and statistical significance was assessed using the non-parametric Kruskal–Wallis test. Dose-response curves and IC₅₀ values were generated to quantify the antibacterial effect. Brintellix demonstrated the strongest antibacterial activity, significantly reducing growth in all 22 strains and showing the lowest mean IC₅₀ value. Cipralex induced moderate inhibition, while Seropram had the weakest effect. The results show that antidepressants can differentially affect bifidobacterial growth and suggest that SMS-class drugs may exert stronger antimicrobial actions than traditional SSRIs. Further studies are needed to explore the broader implications of antidepressant use on microbiota composition and host health, and to compare different antidepressant classes.

The effect of the dietary inclusion of Hermetia illucens larvae meal on the diversity of the methanogenic archaea in the caecum of laying hens (Hy-line Brown) was investigated using molecular methods. A total of 27 hens, selected equally for slaughter from 162 birds which were divided equally into 3 treatment groups including control group C with a diet containing corn-soybean meal and 2 experimental groups, HI25 and HI50, in which 25% and 50% of the soybean meal protein was replaced by the protein from a Hermetia illucens larvae meal, respectively. At 40 weeks of age, the methanogenic community of caecal content of 9 hens per group was analyzed using a 16S rRNA gene clone library. A total of 108 positive clones, 35 from the control group, 44 from the HI25 group and 29 from the HI50 group, were analyzed by Sanger sequencing. Methanomicrobiales, Methanobacteriales and Methanomassiliicoccales were the main orders found in groups C and HI25. Methanomassiliicoccales was absent in the HI50 group, which was dominated by the order Methanobacteriales. At the species level, Methanobrevibacter woesei was the most prevalent species in all three groups regardless of diet. Some species were found exclusively either in the control group (Methanogenic archaeon CH1270) or in the HI25 group (Methanorbis furvi strain Ag1). Methanogenic diversity was significantly lower in the HI50 group compared to the control and HI25 groups and Methanomassiliicoccaceae archaeon DOK was completely suppressed in HI50 group. Our preliminary results indicate that ingestion of Hermetia illucens larvae meal has considerable effect on the methanogenic community, promoting the abundance of Methanobrevibacter woesei and suppressing Methanomassiliicoccaceae archaeon DOK in the caeca of laying hens.

Diabetes mellitus represents a significant global health problem. The number of people suffering from this metabolic disease is constantly rising and although the incidence is heterogeneous depending on region, country, economic situation, lifestyle, diet and level of medical care, it is increasing worldwide, especially among youths and children, mainly due to lifestyle and environmental changes. The pathogenesis of the two most common subtypes of diabetes mellitus, type 1 (T1DM) and type 2 (T2DM), is substantially different, so each form is characterized by a different causation, etiology, pathophysiology, presentation, and treatment. Research in recent decades increasingly indicates the potential role of the gut microbiome in the initiation, development, and progression of this disease. Intestinal microbes and their fermentation products have an important impact on host metabolism, immune system, nutrient digestion and absorption, gut barrier integrity and protection against pathogens. This review summarizes the current evidence on the changes in gut microbial populations in both types of diabetes mellitus. Attention is focused on changes in the abundance of specific bacterial groups at different taxonomic levels in humans, and microbiome shift is also assessed in relation to geographic location, age, diet and antidiabetic drug. The causal relationship between gut bacteria and diabetes is still unclear, and future studies applying new methodological approaches to a broader range of microorganisms inhabiting the digestive tract are urgently needed. This would not only provide a better understanding of the role of the gut microbiome in this metabolic disease, but also the use of beneficial bacterial species in the form of probiotics for the treatment of diabetes.

In the present study, we examined the influence of the dietary inclusion of black soldier fly (BSF) larvae meal on the diversity and composition of the bacterial community in the caecum of Barbary partridges (Alectoris barbara). A total of 54 partridges were divided equally into three treatment groups. The control group (C) received a diet containing corn-soybean meals and the two experimental groups received diets in which soybean meal protein was partially substituted with BSF larvae meal at proportions of 25% (H25) and 50% (H50). The bacterial community of the caecal samples was analysed in 30 slaughtered animals (10 per group) at 64 days of age. High-throughput sequencing targeting the V4-V5 region of the 16 S rRNA gene was used. Firmicutes were the most abundant phylum in all studied categories. This phylum was dominated by the families Ruminococcaceae and Lachnospiraceae. The caecal microbiota was significantly altered at the genus level. The linear discriminant analysis effect size (LefSe) analysis for the differential taxa abundance revealed several significant dissimilarities between the control group (C) and the groups with 25% and 50% insect meal replacement, with 13 and 20 taxa with significantly different abundances, respectively. Several of these taxa are associated with gut health, fiber fermentation, and metabolic functions, indicating a biological importance of the observed microbial shifts. Compared with the control group, the partridges fed 25% BSF larvae meal had a significantly higher bacterial phylogenetic abundance and richness, which may contribute to improved gut health and a more stable microbial environment. The beta diversity measures revealed that all three groups of animals were significantly spatially separated. The results demonstrated the significant impact of black soldier fly larvae meal on the caecal microbiota of Barbary partridges. The positive influence of the insect meal used was indicated by increased bacterial diversity in the H25 group and increased relative abundance of several potentially beneficial genera in both experimental groups.

BACKGROUND:Retrospective evaluation of the frequency, efficacy, and tolerance of antiviral treatment for chronic viral hepatitis B and C (VHB, VHC) in children in Ostrava. MATERIAL AND METHODS:The sample included 17 children with chronic VHB, aged 3-15 years, treated with conventional interferon alpha in 1993-2008, and 11 children with chronic VHC, aged 5-17 years, of whom six were treated with interferon regimens in 1995-2016 and five used direct-acting antivirals since 2020. RESULTS:In the 1990s, children with chronic VHB prevailed; 12 of them were infected vertically. In the last decade, children with chronic VHC dominated, with vertical transmission found in seven children. All 17 children with chronic VHB, 15 of whom were HBeAg (hepatitis B virus e-antigen-positive), were treated with interferon alpha (IFN). HBeAg positivity disappeared in seven children during treatment or within a year after treatment and in another six patients within 15 years after treatment. Treatment tolerance was good, except for psychological problems in two children. Thirteen patients continued to be observed into adulthood, six of whom were treated with interferons or oral antivirals. HBsAg (hepatitis B virus s-antigen) disappeared in four patients aged 7-38 years. Six children with chronic VHC were treated with IFN or pegylated IFN; four received ribavirin. The virus was permanently cleared in five children, the last of whom after treatment with elbasvir and grazoprevir at the age of 18. The tolerance of IFN treatment was good, except for a 5-year-old boy who developed hypothyroidism. Direct-acting antivirals were administered to five children; the first received glecaprevir with pibrentasvir, and the other four received sofosbuvir with velpatasvir. All treatments were successful. Long-term follow-up after treatment was recommended for only three patients. CONCLUSIONS:Antiviral treatment for chronic VHB in children has become a thing of the past thanks to VHB vaccination. However, chronic VHB persists into adulthood after infection in childhood. Currently, chronic VHC dominates in children. After successful treatment with direct-acting antivirals, further follow-up in adulthood is usually unnecessary.

BACKGROUND:The poultry industry is increasingly looking for sustainable feed ingredients which support animal productivity. Insects, such as the larvae of Hermetia illucens, represent a promising alternative protein source that has a positive effect on gut health. AIM:The aim of this study is to evaluate the effects of a partial replacement of soybean meal with H. illucens larvae meal on the caecal microbiota of laying hens. METHODOLOGY:A total of 162 hens were divided equally into three treatment groups: a control group (C) with a diet containing corn-soybean meal and two treatment groups (HI25, HI50) in which 25% and 50% of the soybean meal protein was replaced by H. illucens larvae meal protein. At 40 weeks of age, 30 animals (10 per group) were slaughtered and the bacterial community of the caecal content was analysed by high-throughput sequencing using the V4-V5 region of the 16S rRNA gene. The DNA was extracted using the PowerSoil DNA Kit, the library preparation was performed using the NEBNext Fast DNA Library Prep Set kit and sequencing was performed using the Ion Torrent PGM. The bacterial diversity was assessed by alpha and beta diversity indices, and the differential abundance of taxa was determined using LEfSe analysis. RESULTS:Firmicutes and Bacteroidetes were the dominant phylum in all groups. Alpha diversity indices showed no significant differences between diets, however, beta diversity measures showed statistical dissimilarities between the three studied groups. Several beneficial genera, including Alistipes, Christensenellaceae R-7 group, Parabacteroides, Butyricimonas and Parasutterella, were enriched in the HI50 group, while Lactobacillus, Bifidobacterium, Blautia and the opportunistic pathogen Enterococcus were reduced. CONCLUSION:The consumption of H. illucens larvae meal showed beneficial effect on the microbiota of the caecum of laying hens, with 50% replacement showing the strongest positive effects, suggesting that this is the most effective amount under the given conditions. Further research should explore microbial functions and long-term impacts, and validate the optimal levels of insect meal inclusion.

Preen gland bacteria are thought to be the key producers of preen oil components such as chemosignalling molecules including volatile organic compounds (VOCs) and antimicrobial compounds including peptides and antimicrobial VOCs. However, data on the preen oil bacteriome and chemical composition are limited to a small subset of bird species, and the presence of antimicrobial peptides is largely unexplored. Here, we performed an exploratory study to characterize, for the first time, the preen oil chemical and proteomic profiles and to explore the possible contribution of the bacteriome to the production of preen oil VOCs and antimicrobial peptides (bacteriocins) in eight passerine species, each represented by a single individual. Preen oil bacteriome, chemical and proteomic profiles varied among birds. The bacterial profiles were dominated by the genera Streptococcus, Lactococcus, Corynebacterium and Cutibacterium. The chemical profiles mainly consisted of alcohols, ketones and carboxylic acids. The biological functions primarily associated with the proteomic profiles were proteolysis and response to oxidative stress. Although we were unable to explore a direct association between the bacteriome and chemical profiles, the preen oil contained bacteriocin- and VOC-producing bacterial genera capable of producing detected microbially-derived VOCs (mVOCs), the relative abundance of which varied between birds. Riparian species showed the highest chemical diversity and high abundances of putative preen oil mVOC-producing bacteria, which could suggest habitat-specific adaptations. This exploratory study may significantly contribute to the formulation of hypotheses on the potential role of host ecological factors in the variation of preen oil bacterial, chemical and proteomic profiles in passerines.

Diabetic foot infections (DFIs) contribute to the global disability burden. Beta-lactams are the most commonly used antibiotics for treating DFIs. However, the use of antibiotics may lead to disruption of the healthy balance of the gut microbiota, causing dysbiosis. Patients with infected diabetic foot ulcers (iDFUs) were treated with two kinds of beta-lactams (amoxicillin/clavulanic acid or ceftazidime) according to microbial sensitivity of causative agents via bolus or continuous administration modes. Changes in the gut microbiome of patients were analyzed. Diabetic patients without iDFUs were used as a control group. 16 S ribosomal RNA gene amplicon sequencing was performed on stool samples collected from participants. Alpha diversity and beta diversity of gut microbiota of treated patients did not show significant differences between bolus and continuous modes. However, significant differences were observed between gut microbiota diversity of treated patients and control group. PCoA plots showed individualized responses of the patient’s gut microbiota to antibiotics at different times using both administration forms associated with the pre-treatment state of microbiota composition. Enterococcus, Sellimonas, and Lachnoclostridium were the common bacterial markers differentially abundant in the gut microbiota of antibiotic-treated patients with iDFUs while Roseburia, Dorea, and Monoglobus were mainly abundant in the gut microbiota of patients without iDFUs. Predicted pathways like “Transporters”, “ABC transporters” and “Phosphotranspherase system (PTS)” were upregulated in the gut microbiome of patients treated with bolus regime which may lead to increased intestinal barrier permeability. The present study reported alterations in gut microbiota composition and functionality and provided the bacterial markers as well as potential metabolic signatures associated with each administration mode in patients with iDFUs, which may be used as a reference set for future studies of the effect of antibiotics administration on the gut microbiome of patients with iDFUs. This study shed light on the importance of understanding the effect of antibiotic administration form on gut microbiome in patients with iDFUs. The DFIATIM Clinical Trial (Full title: “Rationalisation of ATB therapy in diabetic foot infection and its impact on the intestinal microbiota”) is submitted to the European Union Clinical Trials Database under the EudraCT Number: 2019-001997-27. The date of registration is July 17th, 2020.

Recently, the bidirectional connection between the gastrointestinal microbiota and the brain has gained interest in many research studies. Findings have highlighted the potential role of stress and sex hormones in modulating the gut microbiome. To our knowledge, no study has investigated the effect of sex hormone perturbations on the gut microbiota in response to stress. To understand how stress may alter the gut microbiota differently depending on sex, gonadectomized and sham-operated male and female mice were subjected to 2 h of daily restraint stress for seven consecutive days. Body weight and plasma level of corticosterone were evaluated. Bacterial diversity and composition of colon and cecum were analyzed by sequencing of 16S rRNA gene. The bacterial communities were strongly altered by stress in the colon than in the cecum. A profound dysregulation of several metabolic and functional pathways was observed in sham mice. Alterations in the gut microbiome diversity and its functional pathways due to stress were more pronounced in males than in females. The present results provide potential sex-specific biomarkers and novel metabolic signatures in the gut microbiota related to stress disorders which may be used as potential targets in diagnostic and therapeutic approaches in neurogastroenterological diseases.

Southern Tamanduas (Tamandua tetradactyla) belong to the specialized placental myrmecophages. There is not much information about their intestinal microbiome. Moreover, due to their food specialization, it is difficult to create an adequate diet under breeding conditions. Therefore, we used 16S rDNA amplicon sequencing to analyze the fecal microbiome of captive Southern Tamanduas from four locations in the Czech Republic and evaluated the impact of the incoming diet and facility conditions on microbiome composition. Together with the microbiome analysis, we also quantified and identified cultivable commensals. The anteater fecal microbiome was dominated by the phyla Bacillota and Bacteroidota, while Pseudomonadota, Spirochaetota, and Actinobacteriota were less abundant. At the taxonomic family level, Lachnospiraceae, Prevotellaceae, Bacteroidaceae, Oscillospiraceae, Erysipelotrichaceae, Spirochaetaceae, Ruminococcaceae, Leuconostocaceae, and Streptococcaceae were mainly represented in the fecal microbiome of animals from all locations. Interestingly, Lactobacillaceae dominated in the location with a zoo-made diet. These animals also had significantly lower diversity of gut microbiome in comparison with animals from other locations fed mainly with a complete commercial diet. Moreover, captive conditions of analyzed anteater included other factors such as the enrichment of the diet with insect-based products, probiotic interventions, the presence of other animals in the exposure, which can potentially affect the composition of the microbiome and cultivable microbes. In total, 63 bacterial species from beneficial commensal to opportunistic pathogen were isolated and identified using MALDI-TOF MS in the set of more than one thousand selected isolates. Half of the detected species were present in the fecal microbiota of most animals, the rest varied across animals and locations.

Retroviruses integrate their genomes into the genomes of infected host cells and form a genetic platform for stable gene expression. Epigenetic silencing can, however, hamper the expression of integrated provirus. As gammaretroviruses (γRVs) preferentially integrate into sites of active promoters and enhancers, the high expression activity of γRVs can be attributed to the integration preference. Long terminal repeats (LTRs) of some γRVs were shown to act as potent promoters for gene expression. Here, we investigate the capacity of different γRV LTRs to drive stable expression inside a non-preferred epigenomic environment using diverse retroviral vectors and CRISPR-Cas9-directed vector knock-in. We demonstrate that different γRV LTRs are either rapidly silenced or long-term active with active proviral population prevailing under normal and retargeted integration. In addition, we show that lamina-associated domains (LADs) can be targeted by CRISPR-Cas9 for vector insertion leading to γRV LTR-driven long-term stable gene expression. Alternatively to established γRV systems, the LTRs of feline leukemia virus and koala retrovirus are capable of driving stable, albeit intensity-diverse, transgene expression in LADs. Altogether, we show that despite the occurrence of rapid silencing events, the majority of γRV LTRs can drive stable expression after retrovirus integration or CRISPR-Cas9-directed knock-in outside of the preferred chromatin landscape. ### Competing Interest Statement The authors have declared no competing interest.

Cutaneous leishmaniasis, a parasitic disease caused by Leishmania major , is a widely frequent form in humans. To explore the importance of the host gut microbiota and to investigate its changes during L. major infection, two different groups of mouse models were assessed. The microbiome of two parts of the host gut—ileum and colon—from infected and non-infected mice were characterised by sequencing of 16S rDNA using an Ion Torrent PGM platform. Microbiome analysis was performed to reveal changes related to the susceptibility and the genetics of mice strains in two different gut compartments and to compare the results between infected and non-infected mice. The results showed that Leishmania infection affects mainly the ileum microbiota, whereas the colon bacterial community was more stable. Different biomarkers were determined in the gut microbiota of infected resistant mice and infected susceptible mice using LEfSe analysis. Lactobacillaceae was associated with resistance in the colon microbiota of all resistant mice strains infected with L. major . Genes related to xenobiotic biodegradation and metabolism and amino acid metabolism were primarily enriched in the small intestine microbiome of resistant strains, while genes associated with carbohydrate metabolism and glycan biosynthesis and metabolism were most abundant in the gut microbiome of the infected susceptible mice. These results should improve our understanding of host-parasite interaction and provide important insights into the effect of leishmaniasis on the gut microbiota. Also, this study highlights the role of host genetic variation in shaping the diversity and composition of the gut microbiome. Key points • Leishmaniasis may affect mainly the ileum microbiota while colon microbiota was more stable. • Biomarkers related with resistance or susceptibility were determined in the gut microbiota of mice. • Several pathways were predicted to be upregulated in the gut microbiota of resistant or susceptible mice. Graphical Abstract

In this study, we investigated the influence of the inclusion of Tenebrio molitor (TM) larvae meal in the diet on the diversity and structure of the bacterial community in the caecal content of Barbary partridges. A total of 36 partridges, selected randomly for slaughter from 54 animals, were divided equally into three treatment groups, including the control group (C) with a diet containing corn-soybean meal and two experimental groups, in which 25% (TM25) and 50% (TM50) of the soybean meal protein was replaced by the meal from TM larvae. After slaughtering, the bacterial community of the 30 caecal samples (10 samples per each experimental group) was analysed by high-throughput sequencing using the V4-V5 region of the 16 S rRNA gene. Alpha diversity showed a higher diversity richness in the TM50 group. Beta diversity showed statistical dissimilarities among the three groups. Firmicutes was the dominant phylum regardless of the diet, with the predominant families Ruminococcaceae and Lachnospiraceae. Clostridia and Faecalibacterium were decreased in both TM groups, Lachnospiraceae was suppressed in the TM50 group, but still this class, genus and family were abundantly present in all samples. Several potentially beneficial genera, such as Bacillus, Ruminococcaceae UCG-009, Oscillibacter and UC1-2E3 (Lachnospiraceae) were increased in the TM50 group. The results showed a beneficial effect of the T. molitor larvae meal on the caecal microbiota of Barbary partridges, particularly in the TM50 group, which showed an increase in bacterial diversity.

Probiotics are a potential strategy for salmonellosis control. A defined pig microbiota (DPM) mixture of nine bacterial strains previously exhibited probiotic and anti-Salmonella properties in vitro. Therefore, we evaluated its gut colonization ability and protection effect against S. typhimurium LT2-induced infection in the gnotobiotic piglet model. The DPM mixture successfully colonized the piglet gut and was stable and safe until the end of the experiment. The colon was inhabited by about 9 log CFU g−1 with a significant representation of bifidobacteria and lactobacilli compared to ileal levels around 7–8 log CFU g−1. Spore-forming clostridia and bacilli seemed to inhabit the environment only temporarily. The bacterial consortium contributed to the colonization of the gut at an entire length. The amplicon profile analysis supported the cultivation trend with a considerable representation of lactobacilli with bacilli in the ileum and bifidobacteria with clostridia in the colon. Although there was no significant Salmonella-positive elimination, it seems that the administered bacteria conferred the protection of infected piglets because of the slowed delayed infection manifestation without translocations of Salmonella cells to the blood circulation. Due to its colonization stability and potential protective anti-Salmonella traits, the DPM mixture has promising potential in pig production applications. However, advanced immunological tests are needed.

Retroviruses integrate into the genomes of infected host cells to form proviruses, a genetic platform for stable viral gene expression. Epigenetic silencing can, however, hamper proviral transcriptional activity. As gammaretroviruses (γRVs) preferentially integrate into active promoter and enhancer sites, the high transcriptional activity of γRVs can be attributed to this integration preference. In addition, long terminal repeats (LTRs) of some γRVs were shown to act as potent promoters by themselves. Here, we investigate the capacity of different γRV LTRs to drive stable expression within a non-preferred epigenomic environment in the context of diverse retroviral vectors. We demonstrate that different γRV LTRs are either rapidly silenced or remain active for long periods of time with a predominantly active proviral population under normal and retargeted integration. As an alternative to the established γRV systems, the feline leukemia virus and koala retrovirus LTRs are able to drive stable, albeit intensity-diverse, transgene expression. Overall, we show that despite the occurrence of rapid silencing events, most γRV LTRs can drive stable expression outside of their preferred chromatin landscape after retrovirus integrations.