Peeling back the layers of competitive exclusion using Salmonella as a model

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. Baby chicks administered a fecal transplant from adult chickens are resistant to Salmonella colonization by competitive exclusion. A two-pronged approach was used to investigate the mechanism of this process. First, Salmonella response to an exclusive (Salmonella competitive exclusion product, Aviguard®) or permissive microbial community (chicken cecal contents from colonized birds containing 7.85 Log10 Salmonella genomes/gram) was assessed ex vivo using a S. Typhimurium reporter strain with fluorescent YFP and CFP genes fusions to rrn and hilA operon, respectively. Second, cecal transcriptome analysis was used to assess the cecal communities’ response to Salmonella in chickens with low (<5.85 Log10 genomes/g) or high (>6.00 Log10 genomes/g) Salmonella colonization. The ex vivo experiment revealed reduction in Salmonella growth and hilA expression following co-culture with the exclusive community. The exclusive community also repressed Salmonella’s SPI-1 virulence genes, LPS modification, while the anti-virulence/inflammatory gene avrA was upregulated. Salmonella transcriptome analysis revealed significant metabolic disparities in Salmonella grown with the two different communities. Propanediol utilization and vitamin B12 synthesis was central to Salmonella metabolism co-cultured with either communities; and mutations in propanediol and vitamin B12 metabolism altered Salmonella growth with the exclusive community. There were significant differences in the cecal community, stress response to Salmonella colonization. Cecal community transcripts indicated that antimicrobials were central to the type of stress response detected in the low Salmonella abundance community suggesting antagonism involved in Salmonella exclusion. This study indicates complex community interactions that modulate Salmonella metabolism and pathogenic behavior and reduce growth by antagonism may be key to exclusion. A six-chip / eleven-sample study using total RNA recovered from Salmonella enterica sv Typhimurium SL1344-derivative YC1104 grown inside dialysis tubing either without any microflora (CTRL), with commercial competitive exclusion product Aviguard outside of the tubing (AV), or with cecal content with high Salmonella load (CECAL35-4) outside of the tubing. Each chip measures the expression level of all Salmonella enterica genes on an oligo array consisting of roughly 380,000 oligos, designed based on the Salmonella typhimurium LT2 (NC_003197.1) and 14028s (CP001363.1: complete genome and CP001362.1: plasmid) genomes, with a moving window of about 12 bases.