Gnotobiotic zebrafish microbiota display inter-individual variability affecting host physiology.

Gnotobiotic animal models reconventionalized under controlled laboratory conditions with multi-species bacterial communities are commonly used to study host-microbiota interactions under presumably more reproducible conditions than conventional animals. The usefulness of these models is however limited by inter-animal variability in bacterial colonization and our general lack of understanding of the inter-individual fluctuation and spatio-temporal dynamics of microbiota assemblies at the micron to millimeter scale. Here, we show underreported variability in gnotobiotic models by analyzing differences in gut colonization efficiency, bacterial composition, and host intestinal mucus production between conventional and gnotobiotic zebrafish larvae re-conventionalized with a mix of 9 bacteria isolated from conventional microbiota. Despite similar bacterial community composition, we observed high variability in the spatial distribution of bacteria along the intestinal tract in the reconventionalized model. We also observed that, whereas bacteria abundance and intestinal mucus per fish were not correlated, reconventionalized fish had lower intestinal mucus compared to conventional animals, indicating that the stimulation of mucus production depends on the microbiota composition. Our findings, therefore, suggest that variable colonization phenotypes affect host physiology and impact the reproducibility of experimental outcomes in studies that use gnotobiotic animals. This work provides insights into the heterogeneity of gnotobiotic models and the need to accurately assess re-conventionalization for reproducibility in host-microbiota studies.

The Impact of Long-Term Macrolide Exposure on the Gut Microbiome and Its Implications for Metabolic Control.

Long-term low-dose macrolide therapy is now widely used in the treatment of chronic respiratory diseases for its immune-modulating effects, although the antimicrobial properties of macrolides can also have collateral impacts on the gut microbiome. We investigated whether such treatment altered intestinal commensal microbiology and whether any such changes affected systemic immune and metabolic regulation. In healthy adults exposed to 4 weeks of low-dose erythromycin or azithromycin, as used clinically, we observed consistent shifts in gut microbiome composition, with a reduction in microbial capacity related to carbohydrate metabolism and short-chain fatty acid biosynthesis. These changes were accompanied by alterations in systemic biomarkers relating to immune (interleukin 5 [IL-5], IL-10, monocyte chemoattractant protein 1 [MCP-1]) and metabolic (serotonin [5-HT], C-peptide) homeostasis. Transplantation of erythromycin-exposed murine microbiota into germ-free mice demonstrated that changes in metabolic homeostasis and gastrointestinal motility, but not systemic immune regulation, resulted from changes in intestinal microbiology caused by macrolide treatment. Our findings highlight the potential for long-term low-dose macrolide therapy to influence host physiology via alteration of the gut microbiome. IMPORTANCE Long-term macrolide therapy is widely used in chronic respiratory diseases although its antibacterial activity can also affect the gut microbiota, a key regulator of host physiology. Macrolide-associated studies on the gut microbiota have been limited to short antibiotic courses and have not examined its consequences for host immune and metabolic regulation. This study revealed that long-term macrolides depleted keystone bacteria and impacted host regulation, mediated directly by macrolide activity or indirectly by alterations to the gut microbiota. Understanding these macrolide-associated mechanisms will contribute to identifying the risk of long-term exposure and highlights the importance of targeted therapy for maintenance of the gut microbiota.

Bacterial host interaction of GFP-labelled Vibrio anguillarum HI-610 with gnotobiotic sea bass, Dicentrarchus labrax (L.), larvae.

The location and cell damage caused by Vibrio anguillarum, the causative agent of classical vibriosis, within the developing gut of the newly hatched sea bass, Dicentrarchus labrax (L.), is unknown. A gnotobiotic sea bass model was used to investigate the early interactions of V. anguillarum with sea bass larvae. In the present study, germ-free sea bass larvae were orally exposed to a V. anguillarum HI-610 pathogen labelled with the green fluorescent protein (GFP-HI-610) and sampled at regular intervals. Pathogenic colonization of gut enterocytes was observed 2 h post-exposure (p.e.) and onwards, whereas bacteria within the swim bladder were visualized 48 h p.e and onwards. Ultrastructural findings demonstrated direct bacterial contact with the host cell in the oesophageal mucosa and putative attachment to microvilli of mid- and hindgut enterocytes. The present findings form a starting point for studies assessing the impact of potential candidates (probiotics, prebiotics, antimicrobial peptides) to mitigate bacterial virulence.

Mechanisms of Kwashiorkor-Associated Immune Suppression: Insights From Human, Mouse, and Pig Studies.

Malnutrition refers to inadequate energy and/or nutrient intake. Malnutrition exhibits a bidirectional relationship with infections whereby malnutrition increases risk of infections that further aggravates malnutrition. Severe malnutrition (SM) is the main cause of secondary immune deficiency and mortality among children in developing countries. SM can manifest as marasmus (non-edematous), observed most often (68.6% of all malnutrition cases), kwashiorkor (edematous), detected in 23.8% of cases, and marasmic kwashiorkor, identified in ~7.6% of SM cases. Marasmus and kwashiorkor occur due to calorie-energy and protein-calorie deficiency (PCD), respectively. Kwashiorkor and marasmic kwashiorkor present with reduced protein levels, protein catabolism rates, and altered levels of micronutrients leading to uncontrolled oxidative stress, exhaustion of anaerobic commensals, and proliferation of pathobionts. Due to these alterations, kwashiorkor children present with profoundly impaired immune function, compromised intestinal barrier, and secondary micronutrient deficiencies. Kwashiorkor-induced alterations contribute to growth stunting and reduced efficacy of oral vaccines. SM is treated with antibiotics and ready-to-use therapeutic foods with variable efficacy. Kwashiorkor has been extensively investigated in gnotobiotic (Gn) mice and piglet models to understand its multiple immediate and long-term effects on children health. Due to numerous physiological and immunological similarities between pigs and humans, pig represents a highly relevant model to study kwashiorkor pathophysiology and immunology. Here we summarize the impact of kwashiorkor on children's health, immunity, and gut functions and review the relevant findings from human and animal studies. We also discuss the reciprocal interactions between PCD and rotavirus-a highly prevalent enteric childhood pathogen due to which pathogenesis and immunity are affected by childhood SM.

Prophylactic Administration of a Bacteriophage Cocktail Is Safe and Effective in Reducing Salmonella enterica Serovar Typhimurium Burden in Vivo.

Nontyphoidal Salmonella bacteria are the causative agent of salmonellosis, which accounts for the majority of foodborne illness of bacterial etiology in humans. Here, we demonstrate the safety and efficacy of the prophylactic administration of a bacteriophage preparation termed FOP (foodborne outbreak pill), which contains lytic phages targeting Salmonella (SalmoFresh phage cocktail), Shiga toxin-producing Escherichia coli (STEC), and Listeria monocytogenes, for lowering Salmonella burdens in OMM12 gnotobiotic mice. Prophylactic administration of FOP significantly reduced the levels of Salmonella in feces and in intestinal sections compared to the levels in controls. Moreover, the overall symptoms of the disease were also considerably lessened. Dose-dependent administration of FOP showed that phage amplification reached similarly high levels in less than 48 h independent of dose. In addition, 16S rRNA gene analysis showed that FOP did not alter the intestinal microbiota of healthy OMM12 mice and reduced microbiota perturbations induced by Salmonella. FOP maintained its full potency against Salmonella in comparison to that of SalmoFresh, its Salmonella-targeting component phages alone. Altogether, the data support that preventive administration of FOP may offer a safe and effective approach for reducing the risk of foodborne infections caused by Salmonella and, potentially, other foodborne bacteria (namely, STEC and L. monocytogenes) targeted by the FOP preparation. IMPORTANCE Foodborne bacterial infections cause worldwide economic loss. During an epidemic, the use of antibiotics to slow down the spread of the disease is not recommended because of their side effects on the resident microbiota and the selection of antibiotic-resistant bacteria. Here, we investigated the potential for the prophylactic administration of bacteriophages (viruses infecting bacteria) to reduce the burden of Salmonella in vivo using mice colonized by a synthetic microbiota. We found that the repeated administration of bacteriophages was safe and efficient in lowering the Salmonella burden. Perturbations of the microbiota by the Salmonella infection were also reduced when mice received bacteriophages. Altogether, these data support the use of bacteriophages as a prophylactic intervention to lower the spread of foodborne epidemics.