ABSTRACT
The anaerobic cultivation of fecal microbiota is a promising approach to investigating how gut microbial communities respond to specific intestinal conditions and perturbations. Here, we describe a flexible protocol using 96-deepwell plates to cultivate stool-derived gut microbiota. Our protocol aims to address gaps in high-throughput culturing in an anaerobic chamber. We characterized the influence of the gas phase on the medium chemistry and microbial physiology and introduced a modular medium preparation process to enable the testing of several conditions simultaneously. Furthermore, we identified a medium formulation that maximized the compositional similarity of ex vivo cultures and donor microbiota while limiting the bloom of Enterobacteriaceae. Lastly, we validated the protocol by demonstrating that cultivated fecal microbiota responded similarly to dietary fibers (resistant dextrin, soluble starch) and drugs (ciprofloxacin, 5-fluorouracil) as reported in vivo. This high-throughput cultivation protocol has the potential to facilitate culture-dependent studies, accelerate the discovery of gut microbiota-diet-drug-host interactions, and pave the way to personalized microbiota-centered interventions.
ABSTRACT
Progressive antibiotic resistance is a serious condition adding to the challenges associated with skin wound treatment, and antibacterial wound dressings with alternatives to antibiotics are urgently needed. Cellulose-based membranes are increasingly considered as wound dressings, necessitating further functionalization steps. A bifunctional peptide, combining an antimicrobial peptide (AMP) and a cellulose binding peptide (CBP), is designed. AMPs affect bacteria via multiple modes of action, thereby reducing the evolutionary pressure selecting for antibiotic resistance. The bifunctional peptide is successfully immobilized on cellulose membranes of bacterial origin or electrospun fibers of plant-derived cellulose, with tight control over peptide concentrations (0.2 ± 0.1 to 4.6 ± 1.6 µg mm-2 ). With this approach, new materials with antibacterial activity against Staphylococcus aureus (log4 reduction) and Pseudomonas aeruginosa (log1 reduction) are developed. Furthermore, membranes are cytocompatible in cultures of human fibroblasts. Additionally, a cell adhesive CBP-RGD peptide is designed and immobilized on membranes, inducing a 2.2-fold increased cell spreading compared to pristine cellulose. The versatile concept provides a toolbox for the functionalization of cellulose membranes of different origins and architectures with a broad choice in peptides. Functionalization in tris-buffered saline avoids further purification steps, allowing for translational research and multiple applications outside the field of wound dressings.
