Project description:An ex vivo system was developed to monitor Salmonella growth, virulence (SPI1 expression) and gene expression (measured by microarray) in response to the permissive and exclusive communities. Yellow fluorescent protein (yfp) and cyan fluorescent protein (cfp) variants were fused to the rrn growth-dependent promoter and the hilA operon (SPI-1 cell invasion locus), respectively, in Salmonella. Fluorescence associated with the YFP and CFP reporters was used to monitor Salmonella growth and SPI1 virulence gene expression in co-culture with cecal communities ex vivo. The Salmonella reporter strain was grown in dialysis tubing in a simulated cecal medium, ex vivo cecal contents (EVCC), submerged in permissive or exclusive communities, to enable collection of Salmonella cells for study. Initially, the fluorescent reporters were used to empirically determine the earliest time point at which the exclusive community had the most significant impact on Salmonella growth or virulence expression relative to the permissive community, which was six-hour co-culture of the reporter strain with the communities. Cells were harvested at that time point for gene expression comparisons. Genes within metabolic pathways that were differentially expressed in permissive vs. exclusive communities were subsequently deleted in Salmonella and mutants’ growth dynamics when cocultured with the exclusive community were monitored over 48 hours using a fluorescence plate reader.
Project description:Clarifying mechanisms underlying the selective adhesion of probiotics and competitive exclusion of pathogens in the intestine is a central theme of maintaining host health.
Project description:The objective of this study was to determine if Salmonella colonization of chickens could be reduced through competitive exclusion using a defined community of chicken commensal bacteria. One-day old White Leghorn chicks, hatched on-site, were randomly divided into experimental groups and given an oral gavage of either a defined community of 15 bacterial species (DC), cecal contents (CC), or sterile PBS (control; CT). After one week, birds were euthanized for cecal content collection (pre-Salmonella sample) while the remaining birds were orally gavaged 1 X 10^8 colony forming units (CFU) of Salmonella enterica ser. Heidelberg strain 2813 (SH2813). Bacterial counts for three post-Salmonella timepoints (3, 14, and 28 days post inoculation; dpi) were evaluated. Bacteriological enumeration was performed by plating cecal contents onto Salmonella selective agar to determine CFU/g in each group for all collection days. Cecal contents were also used for 16S amplicon sequencing. Cecal tissue was used for stranded mRNA sequencing (RNA-Seq).
Project description:Envelope-targeting antimicrobials are widely detected in wastewater and engineered water systems, yet their potential to reshape interspecies interactions through membrane-derived metabolites remains poorly understood. Here, we investigated whether sublethal silver nanoparticles (AgNPs) could bias competitive interactions between Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) toward more balanced coexistence. Repeated exposure to 0.5 mg/L AgNPs for 9 days increased the S. aureus/E. coli ratio threefold. In contrast, untreated co-cultures were dominated by E. coli, with 25% of lineages showing competitive exclusion and an E. coli/S. aureus ratio exceeding 10³ . During competition with S. aureus, E. coli activated ethanolamine (EA) utilization in bacterial microcompartments (Eut BMCs), resulting in extracellular accumulation of acetate and ammonium. Physiological assays identified nitrogen, rather than carbon, as the principal growth-promoting currency of this cross-feeding interaction. Deletion of eutBC in AgNP-exposed cocultures abolished the coexistence phenotype and restored an outcome similar to that observed in the absence of AgNP exposure. In simulated eutrophic water, sublethal AgNPs also enhanced S. aureus survival through the E. coli Eut pathway, supporting the environmental relevance of this mechanism. Mass-matched Ag⁺ exposure neither induced EA utilization nor promoted more balanced coexistence. These findings indicate that sublethal envelope-targeting nanomaterials can alter microbial metabolism and competitive relationships in this two-species model, with potential implications for interactions in contaminated water systems.
Project description:We investigated an improved method that combines competitive PCR and microarray techniques. This approach allowed us to quantify specific bacterial groups mounted on DNA chips with accuracy close to that of real-time PCR, despite a measurement at the end point of PCR, and also to estimate the bacterial DNA content in sample DNA.
Project description:Akkermansia muciniphila, a candidate for next-generation probiotics, can be classified into several clades with distinct genomic divergence. However, the global distribution and population structure of clade-typed A. muciniphila remain poorly understood. Here, we report on the predominance of single A. muciniphila clades in humans globally. We found that the predominance of the A. muciniphila clade is dynamically regulated by the balance between inter-clade inhibitory and pre-occupancy effects. A. muciniphila clade II (AmII)-derived extracellular vesicles exhibited a unilateral inhibitory effect on the growth of AmI. Clade-specific IgA responses were induced by each clade. AmII clade-specific IgAs, also induced by extracellular vesicles, facilitate the competitive exclusion of AmI. Our findings help toward the development of personalized intervention strategies for A. muciniphila, considering a subject’s A. muciniphila type.