The three rhesus monkeys exhibited normal food and water intake, activities, and mental state during antibiotic treatment. Diarrhea symptoms appeared on day 2 after antibiotic treatment, which recovered after one week (Table 1). Hematological levels remained normal after 14 d of antibiotic (ABX) treatment (Figure 1). Compared with conventional (Conv) rhesus monkeys, the mean values for white blood cells, neutrophils, lymphocytes, and mean corpuscular hemoglobin concentration (MCHC) were significantly lower in rhesus monkeys treated with antibiotics (Figure 1A, B). Therefore, the body status tended to be stable after 14 d of antibiotic treatment.
Days post-ABX treatment Severity of diarrhea 1 2 3 0 – – – 1 ++ ++ ++ 2 +++ +++ +++ 3 +++ +++ +++ 4 +++ +++ +++ 5 +++ +++ +++ 6 ++ ++ ++ 7 + + + 8 – + – 9 – – – 10 – – – 1, 2, 3 are IDs of rhesus monkeys. Severity of diarrhea was determined by number of defecations, characteristics of feces, and degree of dehydration. +: Mild diarrhea; ++: Diarrhea; +++: Severe diarrhea; –: Normal stool.
Table 1. Antibiotic treatment induces diarrhea symptoms in rhesus monkeys
Antibiotic treatment resulted in a significant reduction in the abundance of commensal bacteria and reorganization of bacterial composition. According to the results of 16S rDNA amplicon sequencing, 538 722 effective sequences were obtained from samples, with an average length of 448 nt. Taxonomic analysis of OTUs resulted in 1, 1, 12, 20, 37, 61, 143, 218, and 324 different categories at the domain, kingdom, phylum, class, order, family, genus, species, and OTU levels, respectively. Based on α diversity analysis, the abundance and diversity of each sample were sufficient to fully describe the composition of the bacteria, and the sequencing depth was higher than 0.99 (Table 2). After antibiotic treatment, the Shannon index decreased significantly, and the number of OTUs decreased by 96.0%–98.6% after 21 d (Table 2). At the phylum level, the intestinal commensal bacteria in untreated rhesus monkeys mainly consisted of Bacteroidetes and Firmicutes (Figure 2A), and dominant bacteria included Prevotella, Lactobacillus, Bacteroides, Lachnospira, Phascolarctobacterium, Faecalibacterium, Ruminococcus, Megasphaera, and Subdoligranulum (Figure 2B). The diversity and abundance of the commensal bacteria decreased significantly after 21 d; only a few Proteobacteria were detected, and most were antibiotic-resistant Escherichia and Shigella (Figure 2B, C). Based on statistical analysis and PCA, we identified a significant difference in the structure of the commensal bacterial community between untreated and antibiotic-treated rhesus monkeys, and the reorganized bacterial structure maintained stability (Figure 2D, E). We next investigated the enterotype according to the clustering of dominant bacterial communities (Arumugam et al., 2011). Results showed that the bacterial communities were most naturally categorized into eight clusters during treatment (Figure 2F), and antibiotic treatment in rhesus monkeys resulted in a change from Prevotella enterotype to Escherichia-Shigella enterotype (Figure 2G). In addition, the diversity and community structure of the intestinal bacteria in the monkey (ID No. 4) treated only with sucrose remained unchanged (Figure 2H).
ID_days post ABX treatment Abundance index Diversity index Coverage Sobs Ace Chao Shannon Simpson 1_D0 284 286.31 286.55 4.175 399 0.045 147 0.999 685 1_D5 28 86.14 41 0.101 25 0.966 94 0.999 573 1_D14 13 18.79 16.33 0.011 107 0.997 727 0.999 874 1_D21 4 5.59 4 0.003 219 0.999 342 0.999 97 2_D0 227 247.43 254 3.398 891 0.112 415 0.999 196 2_D5 8 43.56 11 0.477 874 0.702 516 0.999 877 2_D14 7 15.84 8.5 0.005 4 0.998 908 0.999 918 2_D21 6 6.83 6 0.009 683 0.997 853 0.999 977 3_D0 249 256.00 260.33 3.843 893 0.060 07 0.999 246 3_D5 28 117.74 49 0.190 655 0.926 19 0.999 52 3_D14 32 149.41 95.33 0.027 369 0.994 332 0.999 415 3_D21 10 91.57 17.5 0.013 941 0.996 685 0.999 844 Data were divided as individuals described in the text. Number of alpha diversity indices are shown.
Table 2. Alpha diversity estimators of 16S rDNA amplicon sequencing
To verify the 16S rDNA amplicon sequencing results, we used quantitative RT-PCR to detect the composition and abundance of intestinal bacteria in the three rhesus monkeys. Results showed that the dominant commensal bacteria in untreated rhesus monkeys consisted of Bacteroides, Prevotella, Lactobacillus, and Clostridium (Supplementary Figure S1A–C). We also tested antibiotic resistance by selective agar medium, and found no antibiotic-resistant strain in the normal commensal bacteria. After 3 d of antibiotic treatment, the copies of commensal bacteria per milligram of feces decreased by 10 000 to 100 000 times (Supplementary Figure S2). After 14 d of antibiotic treatment, the abundance of the 16S rRNA gene decreased significantly in the feces, and predominant bacteria were depleted (Supplementary Figure S1D–F). Furthermore, antibiotics induced the rapid rise of resistant Escherichia coli and Shigella, which did not exist before (Supplementary Figure S1G–I).
After standardization of OTU abundances, the 16S functional predictions were obtained from COG family information according to the corresponding Greengene ID of each OTU. Functional abundances were acquired by analyzing the descriptive and functional information of COG classifications in the eggNOG database. According to the analysis of bacterial functional genomics, we found that the abundance of functional genes in RNA processing and modification, cell motility, and intracellular trafficking were the most significantly increased (P<0.001) after antibiotic treatment (Figure 3A).
To characterize the functional alterations of commensal bacteria in antibiotic-treated rhesus monkeys, we predicted the functional composition profiles from the 16S rDNA sequencing data under PICRUSt pre- and post-antibiotic treatment. We found that multiple KEGG (level 2) categories were disturbed. The changes in pathways enriched in signal transduction, excretory system, neurodegenerative diseases, infectious diseases, and cell motility showed the most significant differences (P<0.001) between untreated and antibiotic-treated rhesus monkeys (Figure 3B). Strikingly, abundances in neurodegenerative disease, infectious disease, cancer, and metabolism pathways were significantly increased after commensal bacteria depletion. In addition, abundances in the digestive system pathway decreased along with signaling molecules and interaction pathways.
As the immune system is modulated by the gut microbiota, we examined whether oral antibiotic treatment affected lymphocyte populations in peripheral blood. We observed increased numbers of CD3+ T cells and CD16+ NK cells after antibiotic treatment, as well as greater CD4+ and CD8+ cells (Figure 4B). In contrast to CD3+ T cells, we observed decreased numbers of Treg cells and CD20+ B cells after antibiotic treatment, suggesting a potential defect in the humoral immune response (Figure 4B). In the cytokine expression profile, we found that CD40L maintained a stable level in serum, indicating a pivotal role in co-stimulation and regulation of the adaptive immune response. The concentration of inflammatory cytokines also varied with the development of intestinal bacterial depletion (Figure 4C).
In addition, we performed an Agilent genome microarray with PBMC samples to acquire gene expression profiling of immune cells. In biological process analysis, we focused on the metabolic process, immune system process, cell communication, and cell activation GO terms. The GO terms of leukocyte activation, immune response, cell-cell signaling, and thyroid hormone metabolic process were significantly different pre- and post-antibiotic treatment (Figure 5). In immune system response and cell-cell signaling, the expression level of LGR4 decreased more than 10-fold. The protein encoded by this gene is a G-protein coulped receptor that binds R-spondins, activates the Wnt signaling pathway, and is associated with osteoporosis (Styrkarsdottir et al., 2013). The IFNA2, CXCL10, and TLR7 genes were also down-regulated, which may affect host susceptibility to viral infection (Karst, 2016; Spurrell et al., 2005; Wu et al., 2013). At the same time, the levels of VCAM1 and IL-13 were upregulated more than 5-fold (Figure 5A, C). It has been reported that IL-13 induces several changes in the gut that can lead to detachment of organisms from the gut wall (Seyfizadeh et al., 2015), and overexpression of IL-13 may contribute to some features of allergic lung diseases such as airway hyperresponsiveness, goblet cell metaplasia, and mucus hypersecretion (Wills-Karp et al., 1998). Several effector factors of immune checkpoint proteins, tissue remodeling, and cell adhesion, such as the MMP14, ABL1, and LILRA6 genes, were markedly elevated. Additionally, several cell communication genes, including GRIA2, BCAN, CACNG3, SLC12A5, SOX17, TDGF1, and VCAM1, were up-regulated (Figure 5C). These results suggest that the absence of commensal bacteria may alter the immune system and disease status of the host.
Compared with the conventional rhesus macaques, the antibiotic-treated monkeys showed impaired morphology of the small intestine. The top of the villi of the intestinal mucosa were diminished, and the epithelial cells of the intestinal mucosa were denatured, necrotic, and shedding, forming extensive superficial erosion (Figure 6). Thus, our results demonstrated that dysbiosis of commensal bacteria could impair the small intestine, as reported in previous animal studies (Kernbauer et al., 2014; Yeruva et al., 2016).
Although drugs are a known cause of neurological symptoms, antibiotics have not been taken seriously and the frequency of severe central nervous system (CNS) events associated with antibiotics is reported to be less than 1% (Bhattacharyya et al., 2016; Owens & Ambrose, 2005). However, the results of a recent retrospective study suggest that bacteria-associated neurogenic disease may be underestimated (Sandler et al., 2000). In our study, one rhesus monkey (ID No. 2) exhibited sudden myoclonus after 20 d of oral antibiotic treatment, which was improved by the reduction of the antibiotic to one third of the designed dose, although convulsive seizure occurred as long as antibiotic treatment continued. Finally, we found abnormal brain morphology in this rhesus monkey. The thalamus appeared congested (Figure 7B), and the area between the inner and outer bundles was sparse, similar to softening foci (Figure 7C).
Health status of rhesus monkeys during oral antibiotic treatment
Antibiotic-treated rhesus monkeys display a dramatic shift in bacterial community structure in the intestine
Predictive functional profiling changes driven by microbial shifts
Impact of commensal bacteria alteration on host immune profile in PBMCs
Depletion of commensal bacteria impairs development of small intestinal morphology
Long-term use of antibiotics leads to severe adverse reactions in a rhesus monkey