Author Contributions

Conceptualization, J.S., Z.W., and E.D.; methodology, Y.X., M.E.M., T.T.; formal analysis, Y.-g.Z.; investigation, Y.X., M.E.M., T.T.; resources, J.S., Z.W., and E.D.; data curation, Y.X., M.E.M., T.T.; writing—original draft preparation, Y.-g.Z., J.S.; writing—review and editing, J.S., Z.W., and E.D., Y.X.; statistical analysis Y.-g.Z., Y.X.; project administration and funding acquisition, J.S., Z.W., and E.D. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the UIC Cancer Center, the NIDDK/National Institutes of Health grant R01 DK105118 and R01DK114126, and DOD CDMRP BC191198 to Jun Sun; Canadian Space Agency grant 18FASASB13 to ED. The contents do not represent the views of the United States Department of Veterans Affairs or the United States Government. This research was also supported by the Office of Naval Research, Naval Research Laboratory base funding (Naval Innovative Science and Engineering (NISE) 1X62, Z.W.). The opinions and assertions contained herein are those of the authors and are not to be construed as those of the U.S. Navy or military service at large. The study sponsors played no role in the study design, data collection, analysis, and interpretation of data.

Figure 1. Gut microbes in the soluble melanin-treated CD-1 mice. The compositions of Escherichia coli and Lactobacillus did not change post 12 and 24 h after CD-1 mice treated with melanin (2.5 mg/mouse), compared to the untreated mice group. Data are expressed as mean ± SD. N = 6, two-way ANOVA test and the p-values were adjusted with Tukey method for multiple comparisons.

Figure 1. Gut microbes in the soluble melanin-treated CD-1 mice. The compositions of Escherichia coli and Lactobacillus did not change post 12 and 24 h after CD-1 mice treated with melanin (2.5 mg/mouse), compared to the untreated mice group. Data are expressed as mean ± SD. N = 6, two-way ANOVA test and the p-values were adjusted with Tukey method for multiple comparisons.

Figure 2. CD-1 mice were one-gavaged with probiotic E. coli Nissle or melanized E. coli Nissle. (A) Relative body weight did not change in CD-1 mice gavaged with probiotic E. coli Nissle or melanized E. coli Nissle. Data are expressed as mean ± SD. N = 6, two-way ANOVA test and the p-values were adjusted with Tukey method for multiple comparisons. (B) The spleen and liver weight did not change significantly in CD-1 mice gavaged with probiotic E. coli Nissle or melanized E. coli Nissle post 48 h. Data are expressed as mean ± SD. N = 6, Welch’s t test. (C) The length of small intestine, cecum and colon did not change in CD-1 mice gavaged with probiotic E. coli Nissle or melanized E. coli Nissle post 48 h. Data are expressed as mean ± SD. N = 6, Welch’s t test. (D) The melanized E. coli Nissle in feces post one-gavage was tested by culture and PCR. The melanized E. coli Nissle (LB cm agar plates, 10 µg/mL) was cultured using one-gavaged mice fecal samples. The melanized bacteria could be detected at 3, 12, and 24 h post treatment. The number of melanized bacteria increased at 3, 12, and 24 h post treatment compared to the before treatment in CFU count. The E. coli 16S rRNA RT-PCR amplification indicates the increased E. coli and melanized E. coli Nissle in fecal samples. Data are expressed as mean ± SD, N = 6, one-way ANOVA test and the p-values were adjusted with Tukey method for multiple comparisons. All p values are shown in this figure. (E) The compositions of Salmonella, Lactobacillus and Bacteroides Fragilis did not change in the melanized bacteria-treated mice. Data are expressed as mean ± SD. N = 6, one-way ANOVA test and the p-values were adjusted with Tukey method for multiple comparisons.

Figure 2. CD-1 mice were one-gavaged with probiotic E. coli Nissle or melanized E. coli Nissle. (A) Relative body weight did not change in CD-1 mice gavaged with probiotic E. coli Nissle or melanized E. coli Nissle. Data are expressed as mean ± SD. N = 6, two-way ANOVA test and the p-values were adjusted with Tukey method for multiple comparisons. (B) The spleen and liver weight did not change significantly in CD-1 mice gavaged with probiotic E. coli Nissle or melanized E. coli Nissle post 48 h. Data are expressed as mean ± SD. N = 6, Welch’s t test. (C) The length of small intestine, cecum and colon did not change in CD-1 mice gavaged with probiotic E. coli Nissle or melanized E. coli Nissle post 48 h. Data are expressed as mean ± SD. N = 6, Welch’s t test. (D) The melanized E. coli Nissle in feces post one-gavage was tested by culture and PCR. The melanized E. coli Nissle (LB cm agar plates, 10 µg/mL) was cultured using one-gavaged mice fecal samples. The melanized bacteria could be detected at 3, 12, and 24 h post treatment. The number of melanized bacteria increased at 3, 12, and 24 h post treatment compared to the before treatment in CFU count. The E. coli 16S rRNA RT-PCR amplification indicates the increased E. coli and melanized E. coli Nissle in fecal samples. Data are expressed as mean ± SD, N = 6, one-way ANOVA test and the p-values were adjusted with Tukey method for multiple comparisons. All p values are shown in this figure. (E) The compositions of Salmonella, Lactobacillus and Bacteroides Fragilis did not change in the melanized bacteria-treated mice. Data are expressed as mean ± SD. N = 6, one-way ANOVA test and the p-values were adjusted with Tukey method for multiple comparisons.

Figure 3. The morphology of colon and ileum did not change in the CD-1 mice one-gavaged with probiotic E. coli Nissle or melanized E. coli Nissle in H&E staining. (A,B) Representative H&E images were from a single experiment and were representative of 6 mice per group.

Figure 3. The morphology of colon and ileum did not change in the CD-1 mice one-gavaged with probiotic E. coli Nissle or melanized E. coli Nissle in H&E staining. (A,B) Representative H&E images were from a single experiment and were representative of 6 mice per group.

Table 1. Real-time PCR Primers.

Table 1. Real-time PCR Primers.

Primers NameSequenceE.Coli F5′-CCTACGGGAGGCAGCAGT-3′E.Coli R5′-CGTTTACGGCGTGGACTAC-3′Salmonella F5′-CACAAATCCATCTCTGGA-3′Salmonella R5′-TGTTGTGGTTAATAACCGCA-3′Lactobacillus F5′-AGCAGTAGGGAATCTTCCA-3′Lactobacillus R5′-CACCGCTACACATGGAG-3′Bacteroides fragilis F5′-CTGAACCAGCCAAGTAGCG-3′Bacteroides fragilis R5′-CCGCAAACTTTCACAACTGACTTA-3′Universal bacteria F5′-TCCTACGGGAGGCAGCAGT-3′Universal bacteria R5′-GGACTACCAGGGTATCTAATCCTGTT-3′