FLX and CUMS Induced Depressive-like Behaviors
There were no body weight or sucrose preference differences in the four groups of rats during the experimental period (Figure 1a and b, one-way ANOVA). CUMS induced significant anxiety-like behaviors in the rats (Figure 1c-f). Rats that received CUMS and FLX walked shorter distances (Figure 1c and d), spent less time in the center area of the box (Figure 1e) and had fewer rearing behaviors (Figure 1f) than rats in the control group (p < 0.001, two-way ANOVA, F = 669.0). These results confirm that CUMS and FLX both induce depressive-like behaviors in normal rats. The administration of FLX into CUMS-treated rats, however, insignificantly offset the depressive-like behaviors.
Summary of the Illumina Sequencing Data
Illumina sequencing captured a total of 2,948,698 highly-qualified sequences, with an average length of 253 bp per sequence and average GC content of 53.49% (Supplementary Table 1). All sequences were classified into 5,373 OTUs (1,430 OTUs per sample, Supplementary Table 1), including 4,042, 3,407, 2,804, and 2,916 OTUs in the control, CUMS, CUMS + FLX and control + FLX groups, respectively. The 5,373 OTUs were affiliated with 54 phyla, 122 classes, 186 orders, 343 families, 704 genera, and over 2000 species. In total, 1,754 overlapping OTUs (32.6%) were identified (Supplementary Figure 1).
Diversity Analysis of Gut Microbiota
There were no differences in the estimators of community richness (ACE and Chao1 indices, Figure 2a and b), sequencing depth (Good’s coverage, Figure 2c) and phylogenetic diversity (PD_whole_tree, Figure 2d) of the OTUs. CUMS decreased the community diversity Shannon’s index by 12.93% (p < 0.05, non-parametric Kruskal-Wallis test, Figure 2e) and weakly reduced Simpson’s index (p > 0.05, non-parametric Kruskal-Wallis test, Figure 2f) compared with the control. The UPGMA trees at the phylum level revealed close relationships and the PCoA analysis showed that the sample data were scattered among 3 quadrants (Figure 3a and b). FLX administration clearly decreased beta diversity in the rats receiving CUMS compared with the control group (p < 0.05; non-parametric Kruskal-Wallis test, Figure 3c).
Significance of CUMS and FLX-altered Gut microbiota Abundance
The main dominant bacterial classes across all samples were Clostridia (Firmicutes phylum, 32.29%-38.16%), Bacilli (Firmicutes phylum, 10.31%-24.95%), Bacteroidia (Bacteroidetes phylum, 19.64%-23.51%) and one unidentified_Actinobacteria class (Actinobacteria phylum, 1.86%-5.71%) (Figure 4b and c). Comparative analysis showed that CUMS with or without FLX administration increased the abundance of Bacilli and unidentified_Actinobacteria (q < 0.05, MetaStat test; Figure 4c) relative to the control. No significant differences were observed in the relative abundances of the other bacterial classes.
At the genus level, Lactobacillus (6.82%-21.06%, Firmicutes phylum), Prevotellaceae_Ga6A1_group (1.11%-7.03%, Bacteroidetes phylum), Romboutsia (3.44%-6.04%, Firmicutes phylum) and [Eubacterium]_coprostanoligenes_group (1.15%-4.06%, Firmicutes phylum) were the dominant bacteria (Figure 4d). CUMS with or without FLX administration only resulted in an increased abundance of Lactobacillus (21.06% vs. 6.82%, q < 0.05 by MetaStat test; Figure 4e). FLX alone did not change the abundances of the dominant bacteria. Additionally, the MetaStat test revealed statistically significant differences for 11 other genera with small proportions (< 0.1%) in the relative abundances (p < 0.05; Supplementary Figure 3). This indicates that chronic FLX administration influenced the diversity of the gut bacterial community in the rats.
We further analyzed the taxonomic differences in the samples at the species level. L. intestinalis (Firmicutes phylum, Bacilli class) represented the main species in all samples (3.35%-6.13%), followed by B. animalis (Actinobacteria phylum, 0.16%-2.71%) and Akkermansia muciniphila (Verrucomicrobia phylum, 0.02%-1.65%, Figure 5a and b). The total proportion of species with less than 0.5% abundance was as high as 88.90%-92.65%. MetaStat testing revealed no significant differences in the dominant species among the groups (p > 0.05).
Lefse Analysis for Significant Gut Microbiota
The LEfSe analysis suggested that significant changes in the bacterial community (at class, order, family and genus level) were induced by CUMS and FLX alone or in combination in the experimental rats in comparison with the control. The taxonomic cladogram revealed that the unidentified_Actinobacter class, a Bacilli class and the Lactobacillales order (Bacilli class) were dominant in the CUMS + FLX group. CUMS alone increased the proportions of the Lactobacillus genus (Bacilli class) and the Lactobacillaceae family (Bacilli class). In contrast, FLX alone only increased the proportion of the Staphylococcaceae family (Bacilli class) in the depression-modeled rats (Figure 5c and Supplementary Figure 4). The detailed relative abundance of each significant bacterial community across all the samples based on the LEfSe analysis is shown in Supplementary Figure 4. These results suggest that CUMS, either alone or in combination with FLX, induced significant differences in the gut microbial communities of the rats.