Chahrazed Mekadim

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. Key points: • 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.

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 (CTZ _bolus ), CTZ continuous infusion (CTZ _continuous ), AMC bolus (AMC _bolus ), and AMC continuous infusion (AMC _continuous ). 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 AMC _bolus and 89% of AMC _continuous . In tissue, target attainment was 60% (CTZ _bolus ), 73% (CTZ _continuous ), 79% (AMC _bolus ), and 83% (AMC _continuous ). 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 AMC _bolus and 89% for AMC _continuous ), target attainment in tissue was substantially lower. These findings suggest that although serum pharmacodynamic targets are reliably achieved, tissue exposure remains suboptimal.

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. Graphical Abstract

Background: 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. Methods: 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. Results: 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. Conclusion: 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. Trial registration: 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.

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.

Bird sex determination is fundamental in various ecological and biological studies, although many avian species cannot be sexed visually due to their monomorphic and/or monochromatic appearance. Thus, reliable laboratory methods for sexing are a prerequisite. Most avian nestlings lack sex-related signs, including the Eurasian pygmy owl (Glaucidium passerinum). We performed laboratory sex determination analysis of this species using blood samples of 242 juveniles and nine adults. It relied on the qPCR of the specific intron from the chromo-helicase DNA-binding protein 1 gene. We tested three primer sets, the P2/P8, 2550F/2718R, and CHD1F/CHD1R, commonly used for bird laboratory sexing. The outcomes were displayed on an agarose gel electrophoresis and a plot from melt curve analysis, which had not been previously conducted in Eurasian pygmy owls. We found that only primer set CHD1F/CHD1R proved reliable, as the only one determined sex with one and two band/s and peak/s on the electrophoresis and the melt curve plot for males and females, respectively. The other two primer pairs failed and depicted one band/peak in all specimens regardless of their sex. Therefore, we recommend performing Eurasian pygmy owls’ laboratory sexing by qPCR with CHD1F/CHD1R primers only.

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 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.

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.

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