Therapeutic Effects of Zymomonas mobilis on Experimental DSS-Induced Colitis Mouse Model

Abstract

Zymomonas mobilis, a Gram-negative bacteria observed in some popular beverages, is considered safe and has been studied for its potential therapeutic benefits. In this study, we explored its effects on the inflammatory process, tissue integrity, differential gene expression, and microbiota composition in an experimental dextran sulfate sodium (DSS)-induced colitis model in mice. As a result, Z. mobilis alleviated the symptoms caused by DSS administration, as indicated by reduced weight loss, disease activity index, a significant reduction in the colon weight/length ratio, and histopathological improvement. Also, Z. mobilis could restore the mucosal barrier as well as increase the expression of Muc3 and Ocln genes. An analysis of 16S rRNA sequences showed that Z. mobilis alters gut microbiota, increasing Akkermansia muciniphila abundance and decreasing Escherichia coli. Furthermore, Z. mobilis seems to be involved in potentiating a regulatory phenotype by inducing immunomodulatory genes like Tgfb, Il5, Il10, and Foxp3 and reducing the relative mRNA expression of proinflammatory cytokines TNF, Il6, and Il17. Our data suggest that Z. mobilis could alleviate disease progression and be considered a possible probiotic adjuvant for pathologies of the bowel.

Keywords: DSS-induced colitis; Zymomonas mobilis; immunomodulation; microbiota; probiotics.

Figure 1

Oral administration of Z. mobilis and its effects on DSS-induced colitis in mice. (a). Animal experimental design: groups of six female C57BL/6 mice were used. Those from DSS and DZM groups ingested filtered water containing 3% DSS in the first 5 days for colitis induction. Throughout the experiment, the animals from the ZM and DZM groups were gavaged with Z. mobilis, while the NC and DSS groups were gavaged with saline. On day 11, all animals were euthanized (represented by the red X). (b). The average body weight (g) of the animals was measured during the ten days of the experiment. Data are shown as the mean ± SEM, and each asterisk color represents the group for which there was a significant statistical difference. Statistical analysis was performed using one-way ANOVA with the Tukey test, * p < 0.05 and ** p < 0.01. (c). DAI was calculated over the entire experiment period. Data are shown as the mean ± SEM, and each asterisk color represents the group for which there was a significant statistical difference. Statistical analysis was performed using one-way ANOVA with the Tukey test, * p < 0.05 and ** p < 0.01. (d). Colon weight/length ratio (cm) after euthanasia. Data are shown as the median and SD (n = 6). Statistical analysis was performed using the Mann–Whitney test, * p < 0.05 and ** p < 0.01.

Figure 2

Histopathological analysis and effects of Z. mobilis on mouse colon mucosa. (a). Representative H&E staining of colon tissues in the NC, DSS, ZM, and DZM groups. LP: lamina propria; GC: goblet cell; CC: colon crypts; MM: muscularis mucosae; SM: submucosa; ME: muscularis externa; inflammatory cells increased in the lamina propria (blue arrow); crypt architectural disarray/crypt loss/irregular crypts/crypts with hyperplasia (white arrowhead); erosion (black arrowhead); crypt abscess (yellow arrowhead). Slides were analyzed under 20× (scale bar 100 µm) or 40× (scale bar 50 µm) magnification power with Aperio ImageScope Software (Leica, Wetzlar, Germany). (b). Histological score of colon tissues. Data are shown as means and SD (n = 6). Statistical analysis was performed using the Mann–Whitney test: * p < 0.05 and ** p < 0.01.

Figure 3

Alpha diversity analysis. (a). Observed species in each sample pool on days 5 and 10. (b). Shannon diversity index measured in each sample pool on days 5 and 10. (c). Chao1 richness index measured in each sample pool on days 5 and 10.

Figure 4

Composition of gut microbial community in mice. (a). Histogram of relative abundance of dominant phyla on days 5 and 10. (b). Ternary plots of relative abundance of dominant species on days 5 and 10 in which each corner of the triangle represents a time point (D5, NZW5, DZW5 or D10, NZW10, DZW10) and each circle size represents species abundance.

Figure 5

Microbiome diversity analysis. (a). Heatmap of topmost abundant genera of mice gut microbiota groups NC, DSS, ZM, and DZM on days 5 and 10 of analyzes. (b,c). Venn diagram of OTUs distributed among groups on day 5 and day 10, respectively. (d). PCA plot points represent different sample groups and their clusters.

Figure 6

Colonic mRNA expression by real-time PCR analysis. Relative gene expression: (a). Muc3; (b). Ocln; (c). Foxp3; (d). Tgfb; (e). Il10; (f). Ifng; (g). Il5; (h). Stat6; (i). Tnfa; (j). Il6; (k). Il17; (l). Il1b. Data are shown as means and SD (n = 6). Statistical analysis was performed using the Mann–Whitney test: * p < 0.05 and ** p < 0.01.