Response of Salmonella enterica serovar Typhimurium to alginate oligosaccharides fermented with fecal inoculum: integrated transcriptomic and metabolomic analyses

Abstract

Alginate oligosaccharides (AOS), extracted from marine brown algae, are a common functional feed additive; however, it remains unclear whether they modulate the gut microbiota and microbial metabolites. The response of Salmonella enterica serovar Typhimurium, a common poultry pathogen, to AOS fermented with chicken fecal inocula was investigated using metabolomic and transcriptomic analyses. Single-strain cultivation tests showed that AOS did not directly inhibit the growth of S. Typhimurium. However, when AOS were fermented by chicken fecal microbiota, the supernatant of fermented AOS (F-AOS) exhibited remarkable antibacterial activity against S. Typhimurium, decreasing the abundance ratio of S. Typhimurium in the fecal microbiota from 18.94 to 2.94%. Transcriptomic analyses showed that the 855 differentially expressed genes induced by F-AOS were mainly enriched in porphyrin and chlorophyll metabolism, oxidative phosphorylation, and Salmonella infection-related pathways. RT-qPCR confirmed that F-AOS downregulated key genes involved in flagellar assembly and the type III secretory system of S. Typhimurium, indicating metabolites in F-AOS can influence the growth and metabolism of S. Typhimurium. Metabolomic analyses showed that 205 microbial metabolites were significantly altered in F-AOS. Among them, the increase in indolelactic acid and 3-indolepropionic acid levels were further confirmed using HPLC. This study provides a new perspective for the application of AOS as a feed additive against pathogenic intestinal bacteria.

Supplementary information: The online version contains supplementary material available at 10.1007/s42995-023-00176-z.

Keywords: Alginate oligosaccharides; Gut microbiota; Metabolite; Metabolomics; Salmonella enterica serovar Typhimurium; Transcriptomic.

Fig. 1

Inhibitory effect of AOS against S. Typhimurium during in vitro chicken fecal fermentation. A Total bacterial colony count of gut microbiota. B S. Typhimurium colony count in gut microbiota. C The abundance ratios of S. Typhimurium in the chicken gut microbiota. CK, without adding any oligosaccharides. Statistical significance was determined by Student’s t-test (n = 3). ns, nonsignificant; *, P < 0.05; **, P < 0.01

Fig. 2

Antibacterial activity of AOS and F-AOS during single S. Typhimurium cultivation. A-D, S. Typhimurium was treated with AOS. E, S. Typhimurium was treated with F-AOS. A Growth curves of S. Typhimurium. B Changes of pH values. C The mRNA level of pathogenicity related genes (sipA, invA, and ompW) were assessed using RT-qPCR. D TLC profiles of the utilization of AOS at 0, 48, 96, and 144 h, respectively. E OD₆₀₀ of S. Typhimurium in F-AOS and F-Non-O. CK, control group; M, mannuronate oligosaccharides (DP 1–6); G, guluronate oligosaccharides (DP 2–6); I-VI, DP 1–6; F-AOS, supernatant of AOS fermented with chicken fecal inoculum. F-Non-O, supernatant of fermented non-O. Significant differences among substrates at the same time point were calculated using Student’s t-test (n = 3). ns, nonsignificant; *, P < 0.05; **, P < 0.01

Fig. 3

Effects of F-AOS on S. Typhimurium transcriptomics. A PCA plot. B Volcano graph of 855 DEGs. Red represents upregulated DEGs; green represents downregulated DEGs. F-AOS, supernatant of AOS fermented with chicken fecal inoculum. Supernatant of non-O fermented (F-Non-O) was used as the control

Fig. 4

KEGG and GO analysis. A KEGG pathway classification of genes. B GO classifications of genes. C KEGG pathway enrichment analysis. D GO enrichment analysis. Rich factor, number of differentially expressed genes total number. F-AOS, supernatant of AOS fermented with chicken fecal inoculum. Supernatant of non-O fermented (F-Non-O) was used as the control

Fig. 5

Effects of AOS supplementation (F-AOS) on fecal microbiota metabolomics. The medium without adding any oligosaccharides was used as the control (F-Non-O). A PCA score plot. B OPLS-DA score plot. C The volcano plot was used to visualize differences between groups with yellow dots representing distinct upregulated metabolites, purple dots representing downregulated metabolites, and grey dots representing non-significant differences. D Bubble diagram of enriched KEGG pathways. Sizes of dots represent number of metabolites

Fig. 6

Metabolite analysis of AOS supplementation (F-AOS) on fecal microbiota metabolomics. The medium without the addition of oligosaccharides was used as the control (F-Non-O). A VIP scores analysis. Heatmap with yellow or blue boxes on the left indicates high and low abundance ratio, respectively. B The abundance ratio of selected potential metabolites biomarkers. Statistical significance was determined by Student’s t-test (n = 6). ***, P < 0.001

Fig. 7

Integrated transcriptomic and metabolomic analysis of inhibition effect of F-AOS against S. Typhimurium. A RT-qPCR results of seven DEGs. The expression of each gene was normalized to the average expression of the endogenous reference gene 16S rRNA. B RNA-seq results of the DEGs. Quantification of potential biomarkers of indolelactic acid C, 3-indolepropionic acid D, N-acetylneuraminic acid E, and p-cresol F in F-AOS by HPLC. G Possible responses of S. Typhimurium to F-AOS. Red colors represent downregulated at the mRNA level. F-AOS, supernatant of AOS fermented with chicken fecal inoculum. Supernatant of non-O fermented (F-Non-O) was used as the control. Statistical significance was determined by Student’s t-test (n = 3). **, P < 0.01