Intestinal microorganisms assist the host in digesting complex and difficultly decomposed foods; expand the host’s dietary ecological niche. In order to investigate the effect of high-fiber food on intestinal microorganisms of Eothenomys miletus at different altitudes, exploring the regional differences of intestinal microorganisms and their roles in body mass regulation, we collected E. miletus from Dali (DL) and Xianggelila (XGLL), which were divided into control group, high-fiber group fed with high-fiber diet for 7 days, and refeeding group fed with standard diet for 14 days after high-fiber diet. Using 16S rRNA gene sequencing technology combined with physiological methods, we analyzed the gut microbial diversity, abundance, community structure and related physiological indicators of each group, and explored the effects of high-fiber foods and regions on the diversity, structure of gut microorganisms and physiological indicators. The results showed that high-fiber food affected the food intake and metabolic rate of E. miletus, which also showed regional differences. The intestinal microorganisms of E. miletus obtained energy through the enrichment of fiber degrading bacteria under the condition of high-fiber food, while producing short-chain fatty acids, which participated in processes such as energy metabolism or immune regulation. Moreover, it also affected the colonization of intestinal microorganisms. High-fiber food promoted the enrichment of probiotics in the intestinal microbiota of E. miletus, but pathogenic bacteria also appeared. Therefore, the changes in the composition and diversity of gut microbiota in E. miletus provided important guarantees for their adaptation to high fiber food environments in winter.

In microbiome studies, fecal and oral samples are stored and processed in different ways, which could affect the observed microbiome composition. In this study, we compared storage and processing methods applied to samples prior to DNA extraction to determine how each affected microbial community diversity as assessed by 16S rRNA gene sequencing. We collected dental swabs, saliva, and fecal samples from 10 individuals, with three technical replicates per condition. We assessed four methods of storing and processing fecal samples prior to DNA extraction. We also compared different fractions of thawed saliva and dental samples to fresh samples. We found that lyophilized fecal samples, fresh whole saliva samples, and the supernatant fraction of thawed dental samples had the highest levels of alpha diversity. The supernatant fraction of thawed saliva samples had the second highest evenness compared to fresh saliva samples. Then, we investigated the differences in observed community composition at the domain and phylum levels and identified the amplicon sequence variants (ASVs) that significantly differed in relative abundance between the conditions. Lyophilized fecal samples had a greater prevalence of Archaea as well as a greater ratio of Firmicutes to Bacteroidetes compared to the other conditions. Our results provide practical considerations not only for the selection of storage and processing methods but also for comparing results across studies. Differences in processing and storage methods could be a confounding factor influencing the presence, absence, or differential abundance of microbes reported in conflicting studies.

Antimicrobial resistance (AMR) is a global, multifaceted crisis that poses significant challenges to the successful eradication of devastating pathogens, particularly methicillin-resistant Staphylococcus aureus (MRSA), a persistent superbug that causes devastating infections. The scarcity of new antibacterial drugs is obvious, and antivirulence strategies that reduce the pathogenicity of bacteria by weakening their virulence have become the subject of intense investigation. Alpha-hemolysin (Hla), a cytolytic pore-forming toxin, has a pivotal role in S. aureus pathogenesis. Here, we demonstrated that echinatin, a natural compound isolated from licorice, effectively inhibited the hemolytic activity of MRSA at 32 μg/mL. In addition, echinatin did not interfere with bacterial growth and had no significant cytotoxicity at the inhibitory concentration of S. aureus hemolysis. Heptamer formation tightly correlated with Hla-mediated cell invasion, whereas echinatin did not affect deoxycholic acid-induced oligomerization of Hla. Echinatin affected hemolytic activity through indirect binding to Hla as confirmed by the neutralization assay and cellular thermal shift assay (CETSA). Furthermore, qRT–PCR and western blot analyses revealed that echinatin suppressed Hla expression at both the mRNA and protein levels as well as the transcript levels of Agr quorum-sensing system-related genes. Additionally, when echinatin was added to a coculture system of A549 cells and S. aureus, it significantly reduced cell damage. Importantly, echinatin exhibited a significant therapeutic effect in an MRSA-induced mouse pneumonia model. In conclusion, the present findings demonstrated that echinatin significantly inhibits the hemolysin effect and may be a potential candidate compound for combating drug-resistant MRSA infections.