Jianfei Zhu

Background: Phospholipids are essential components of bacterial membranes and play central roles in membrane integrity and adaptation to antibiotic stress. However, confident annotation of phospholipid molecular species remains challenging due to the complexity of the lipidome and the limited structural constraints in conventional lipidomics workflows. Methods: Here, we present a bacterial phospholipidomic framework that integrates orthogonal structural evidence to achieve high-confidence and traceable annotation. Thin-layer chromatography (TLC) provides phospholipid headgroup assignment, gas chromatography–mass spectrometry (GC–MS) defines the acyl-chain pool, and Paternò–Büchi derivatization enables C=C localization, collectively restricting the structural search space prior to liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis. A rule-based ion prediction library further standardizes diagnostic ion assignment and reduces annotation ambiguity. Results: Applying this platform, we found Escherichia coli in the stationary phase remodeled the membrane phospholipids, with cardiolipin (CL) increasing from ~5% to ~10% and cyclopropane-containing phospholipid species rising to ~75%. Similar remodeling patterns are observed under diverse antibiotic exposures at sub-inhibitory concentrations, consistent with convergence toward a tolerance-associated membrane state. Extension of the framework to Enterococcus faecium supports proof-of-concept application in an additional Gram-positive model, with vancomycin-resistant strains exhibiting pronounced phosphatidylglycerol (PG) enrichment and reduced CL. Conclusions: Our work provides a scalable and reproducible strategy for bacterial phospholipid annotation, enabling molecular-species-resolved investigation of membrane adaptation and offering a framework for future exploration of lipid homeostasis pathways as potential antimicrobial targets.

Background: Salmonella enterica subsp. enterica serovar Gallinarum biovar Pullorum (Salmonella Pullorum) is a specific avian pathogen responsible for Pullorum disease, causing substantial economic losses to the global poultry industry. With the rising restrictions on antibiotic use, probiotics have emerged as promising therapeutic alternatives. The Bacillus subtilis group, including B. amyloliquefaciens and B. subtilis, is a collection of closely related species that has been widely used as a probiotic due to its broad-spectrum antimicrobial activity and other benefits. However, how the probiotics-derived antibacterial phenotype contributes to infection control is still unclear. Methods: In this study, we used two different antibacterial phenotype strains, B. amyloliquefaciens and B. subtilis, to treat S. Pullorum infections. The spores of two strains (10⁷ CFUs) were supplemented daily for 21 days. Results: The reduction in body weight gains and the severity of S. Pullorum-induced symptoms were ameliorated. Compared to B. subtilis, B. amyloliquefaciens exhibited a stronger host protection effect, manifested in a greater reduction in the bacterial load of S. Pullorum in organs throughout the infection. Furthermore, both strains enhanced cecal microbiota diversity, suppressed infection-associated taxa, and promoted beneficial genera. Conclusions: Our findings demonstrate that probiotic Bacillus can alleviate S. Pullorum infection and improve growth performance in poultry, especially the antimicrobial phenotype contributing to pathogen clearance. This work provides crucial insights for developing effective, probiotic-based strategies against Pullorum disease.

Intestinal bacterial infections are a major threat to human and animal health. In this study, we found plant-derived antibacterial xanthones, particularly α-mangostin (AMG) from the mangosteen peel, exhibiting extraordinary activities against Clostridium perfringens. Structure–activity relationship analysis showed that prenylation modulated the activity of xanthones. The efficacy of AMG (4, 8, 20 mg/kg body weight) was also demonstrated in the broiler chicken necrotic enteritis model infected with Clostridium perfringens. In the models (n = 6 per group), feed supplementation of AMG maintained the homeostasis of the gut microbiome by reducing the colonization of clostridia and promoting the integrity of intestinal barriers via the upregulation of mucin expression. These results suggest that plant-derived xanthones may be a potential alternative to antibiotics for treating clostridial enteric infections in the clinic.