Radiation exposure effects include diarrhea due to changes in intestinal microbial flora and damage to the intestinal lining. These changes persist after the cessation of radiation . Hence it is important to understand the profile of changes in the intestinal microbial flora after radiation and to identify biomarkers of radiation-induced diarrhea. Due to the evolutionary proximity to humans, NHP were used in our studies to explore the alteration in the gut microbiome composition after radiation exposure. NHPs (N=19) were exposed to whole body 7.4 Gy cobalt-60 gamma-radiation (LD70-80/60) (Table.1), followed by fecal sample collection and monitoring for clinical signs of adverse effects with weekly body weight measurements. Fecal samples collected before (day -1) and after (day +1 and +4) irradiation were subjected to 16S RNA gene sequencing.
Alpha and beta diversity analysis pre and post-irradiation: 441 to 560 were observed in the fecal samples at pre-irradiation (day -1), whereas 381 to 578 were observed post-radiation (day +4). Diversity of microbes within the samples (Alpha Diversity) was measured by the Shannon Diversity Index, Chao1, and Simpson index (Supplementary Table 2). Shannon index was not significantly different between the pre- and post-irradiation sets of samples (5.96 ± 0.72 vs 5.58 ± 0.76 respectively) (Figure 1). Similarly, other indices (Chao1 and Simpson index) also failed to show any significant difference (Supplementary Table 2).
To compare the diversity in the microbial composition between the fecal samples collected pre and post-irradiation, Beta diversity was measured using three metrics; Bray Curtis (non-phylogeny based) (BC), weighted (W) and unweighted phylogeny based UNIFRAC (UW). While the weighted UNIFRAC (W) analysis measures abundance of the microbiome, unweighted UNIFRAC (UW) does not account for the microbial abundance. As shown in Table 2, fecal samples immediately after irradiation (at day +1) did not show a significant difference compared to pre-irradiation (day -1) samples (p values not significant). In contrast, fecal samples at day +4 post-irradiation showed a significant difference (p values <0.05), suggesting significant differences in the microbe composition after irradiation, in a time dependent fashion.
Analysis of abundance at the Phylum and Genus levels: 16S data base was used to analyze the microbial abundance at the Phylum and Genus levels, shown in Table 3. As there was no significant change in the beta diversity immediately after the irradiation (day +1 post-irradiation) (Table 2), analysis was limited to samples collected later (day +4) in comparison to pre-irradiation (day -1) samples.
Before irradiation (day -1), Bacteriodetes and Firmicutes were the major phyla observed (39 to 46%). Other minor phyla such as Spirochaetes (5.6%), Verrucomicobria (1.2%), and less than 1% of other phyla (Euryarcheaota, Lentisphaerae, Tenericutes) were observed. As noted elsewhere ,, baseline gut microbiota was dominated by members of the Prevotella (30.5%) genus (data not shown). However, after irradiation (Day +4) the prevalence of Bacteroidetes phylum increased from 39% to 52% (1.3-fold increase), whereas Firmicutes decreased from 46% to 39% (0.84-fold decrease), resulting in a significant decrease in the Firmicutes/Bacteriodes (F/B) ratio from 1.2 at pre-irradiation (day -1) to 0.75 at post-irradiation (day +4) (p=0.014).
Representation of four genera, Prevotella (Paraprevotellaceaea family), Actinobacillus, Veillonella and Bacteroides, was found to be increased between 2 and 11 fold, whereas representation of two genera, - Acinetobacter and Aerococcus, decreased by 10 and 100 fold, respectively, at day +4 post-irradiation (Table 3). Considering the prevalence before irradiation (more than 0.1%), three differences at the genus levels at day +4 post-irradiation were noteworthy. These include, increased Prevotella (Paraprevotellaceaea family) (more than 4-fold increase) and decreased Acinetobacter and Aerococcus (more than 10 to 100-fold decreases, respectively).
Microbial changes in animals with diarrhea post-irradiation: Diarrhea was observed in some animals in a time dependent manner as a consequence of irradiation. None of the animals had shown diarrhea pre-irradiation, and day +1 and +2 post-irradiation, whereas 10 out of 19 (52.6%) showed diarrhea at day +4 post-irradiation. By day 6, 79% of the animals came down with diarrhea. As we wanted to study the early microbial changes associated with diarrhea, we focused on animals with diarrhea at day +4. Comparison of the microbiome of animals with and without diarrhea was performed to understand the microbial differences. PCoA plot analysis for the gut microbiome(unweighted UNIFRAC) of animals with and without diarrhea at day +4 post-irradiation in comparison to baseline suggested significant changes in the microbiome population after radiation at day +4 (Figure 3). Detailed analysis of these changes suggested three significant differences in these animals (Table 4). Lactobacillus reuteri was significantly increased (17 fold) in prevalence (0.06% to 1.09%) in animals with diarrhea. Similarly, two genera, Dialister (14.9 fold) and Veillonella (32.9 fold) were also found to be increased in animals with diarrhea compared to animals without diarrhea.
Biomarkers for radiation-induced diarrhea: Diarrhea is a significant side effect of radiation injury . It would be beneficial to prospectively identify subjects who might be prone to radiation induced diarrhea, to develop appropriate therapy. To identify such microbial biomarkers, fecal samples of animals at pre-irradiation (day -1) with and without diarrhea symptom post-irradiation (day +4) were compared (Table 5). Before irradiation (day -1), two phylum of bacteria were associated with diarrhea at day +4. Animals with diarrhea had significantly lower levels of Lentisphaere and Verrucomicrobia phyla and significantly lower levels of Bacteroides genus before irradiation. These differences show the potential association between the prevalence of microbiomes and differential susceptibility to radiation-induced diarrhea.