Perturbation of the small intestine microbial ecology by streptomycin alters pathology in a Salmonella enterica serovar typhimurium murine model of infection

Abstract

The small intestine is an important site of infection for many enteric bacterial pathogens, and murine models, including the streptomycin-treated mouse model of infection, are frequently used to study these infections. The environment of the mouse small intestine and the microbiota with which enteric pathogens are likely to interact, however, have not been well described. Therefore, we compared the microbiota and the concentrations of short-chain fatty acids (SCFAs) present in the ileum and cecum of streptomycin-treated mice and untreated controls. We found that the microbiota in the ileum of untreated mice differed greatly from that of the cecum of the same mice, primarily among families of the phylum Firmicutes. Upon treatment with streptomycin, substantial changes in the microbial composition occurred, with a marked loss of population complexity. Characterization of the metabolic products of the microbiota, the SCFAs, showed that formate was present in the ileum but low or not detectable in the cecum while butyrate was present in the cecum but not the ileum. Treatment with streptomycin altered the SCFAs in the cecum, significantly decreasing the concentration of acetate, propionate, and butyrate. In this work, we also characterized the pathology of Salmonella infection in the ileum. Infection of streptomycin-treated mice with Salmonella was characterized by a significant increase in the relative and absolute levels of the pathogen and was associated with more severe ileal inflammation and pathology. Together these results provide a better understanding of the ileal environment in the mouse and the changes that occur upon streptomycin treatment.

FIG. 1.

The microbial composition of the ileum. 16S rRNA gene clone libraries were created from the ileum and cecum of C57BL/6 mice. (A) Taxonomic analysis of the clone library sequences was performed using RDP Classifier and SeqMatch. Assignment to the phylum level for the sequences from the ileum and cecum of untreated mice is displayed with further distinction among the phylum Firmicutes to the family level. The total of the Firmicutes is shown by the arc extending around the pie chart. (B) Cluster analysis of the Bray-Curtis distance measure of diversity between the microbial communities for the ileum and cecum from untreated mice. For the Bray-Curtis distance measure, an OTU was defined as 97% sequence similarity. (C) 16S rRNA gene clone libraries were created from samples containing ileal tissue and contents or contents alone from untreated mice. Sequences were assigned to OTUs using a definition of 97% sequence similarity. A heat map is used to show the relative abundance of OTUs, with specific OTUs detected in the sample oriented along the horizontal axis, and the dendrogram showing the distribution of OTUs. Darker coloring within the heat map indicates greater representation of specific OTUs. Phyla are shown above the figure, and highly represented OTUs are shown below. Numbers 1 to 6 and 16P to 18P represent individual mice.

FIG. 2.

Streptomycin treatment alters the microbial composition of both the ileum and the cecum. (A) Rarefaction analysis and 95% confidence intervals are shown for the 16S rRNA-encoding sequences obtained from each site in the untreated and streptomycin-treated mice. Central lines represent rarefaction analysis of each site with the surrounding lines representing the upper and lower 95% confidence intervals. For rarefaction analysis, an OTU was defined as 97% sequence similarity. (B) 16S rRNA gene clone libraries were created from samples containing ileal tissue and contents from untreated mice and those pretreated with streptomycin. Sequences were assigned to OTUs using a definition of 97% sequence similarity. A heat map is used to show the relative abundance of OTUs, with specific OTUs detected in the sample oriented along the horizontal axis, and the dendrogram showing the distribution of OTUs. Darker coloring within the heat map indicates greater representation of specific OTUs. Phyla are shown above the figure, and highly represented OTUs are shown below. Untreated mice shown in this figure are the same as those shown in Fig. 1C.

FIG. 3.

Streptomycin pretreatment increases Salmonella infection of the ileum. Real-time PCR was used to compare the numbers of Salmonella bacteria in the ilea of individual mice without additional treatment to those in mice pretreated with oral streptomycin. The number of copies of the Salmonella 16S rRNA gene amplified using Salmonella-specific primers was determined and is shown relative to the mass of the ileal sample. The limit of detection for Salmonella was one copy/100 ng total DNA. Represented adjacent to the circles are the numbers of Salmonella 16S rRNA gene sequences identified over the total size of the clone library analyzed from this same animal. Circles with “nd” (not determined) show animals in which clone libraries could not be produced, primarily due to low bacterial DNA yields. Horizontal lines represent the medians for each group.

FIG. 4.

SCFAs of the untreated and streptomycin-treated ileum and cecum. Intestinal contents were collected from the ileum and cecum of both untreated and streptomycin-treated mice 72 h after treatment. SCFAs were quantified using HPLC analysis for the ileum (A) or GC-MS for the cecum (B). The box plots show the medians with 25% and 75% quartiles. The bars in each box plot extend to the outermost points located within the quartile ± 1.5× the interquartile range. An asterisk indicates a significant difference at a P value of <0.01 between the untreated mice and streptomycin-treated mice for SCFA concentrations.

FIG. 5.

Streptomycin administration prior to infection with Salmonella enhances the extent and degree of ileal inflammation in mice. Hematoxylin- and eosin-stained ileum was obtained from mice pretreated with streptomycin and infected with Salmonella. (A) Necrosis of ileal villi accompanied by focally extensive infiltration of lamina propria by large numbers of polymorphonuclear neutrophils extending along the base of intestinal crypts. Magnification, ×12. (B) Infiltration of villous lamina propria by large numbers of polymorphonuclear neutrophils with segmental sloughing of intestinal epithelial cells. Magnification, ×20. (C) Infiltration of ileal Peyer's patch subepithelial dome area by large numbers of polymorphonuclear neutrophils (asterisk) together with focal disruption of the follicle-associated epithelium (arrowhead) and transepithelial migration of neutrophils. Magnification, ×20.

FIG. 6.

Streptomycin administration prior to infection with Salmonella is associated with ileal Peyer's patch inflammation in mice. Hematoxylin- and eosin-stained ileal Peyer's patches of uninfected mice and mice infected with Salmonella with and without streptomycin pretreatment. (A and B) Untreated and uninfected; (C and D) streptomycin treated; (E and F) Salmonella infected; (G and H) streptomycin treated and Salmonella infected. The subepithelial dome area is diffusely infiltrated by large numbers of polymorphonuclear neutrophils in the streptomycin-treated and Salmonella-infected mouse (G and H) (asterisks), while the follicle-associated epithelium is markedly attenuated in mice infected with Salmonella with (H) (arrowhead) or without (F) (arrowhead) streptomycin compared with tall columnar epithelium in uninfected mice (B and D) (arrowheads). The streptomycin-treated and Salmonella-infected mouse also displays focal disruption of the follicle-associated epithelium along with multifocal transepithelial migration of polymorphonuclear neutrophils and focal crypt abscess (H) (arrow). Magnifications, ×6 (A, C, E, and G) and ×12 (B, D, F, and H).