Influence of Fluconazole Administration on Gut Microbiome, Intestinal Barrier, and Immune Response in Mice

ABSTRACT

Antibiotics that can treat or prevent infectious diseases play an important role in medical therapy. However, the use of antibiotics has potentially negative effects on the health of the host. For example, antibiotics use may affect the host's immune system by altering the gut microbiota. Therefore, the aim of the study was to investigate the influence of antifungal (fluconazole) treatment on the gut microbiota and immune system of mice. Results showed that the gut microbial composition of mice receiving fluconazole treatment was significantly changed after the trial. Fluconazole did not affect the relative abundance of bacteria but significantly reduced the diversity of bacterial flora. In the bacteriome, Firmicutes and Proteobacteria significantly increased, while Bacteroidetes, Deferribacteres, Patescibacteria, and Tenericutes showed a remarkable reduction in the fluconazole-treated group compared with the control group. In the mycobiome, the relative abundance of Ascomycota was significantly decreased and Mucoromycota was significantly increased in the intestine of mice treated with fluconazole compared to the control group. Reverse transcription-quantitative PCR (RT-qPCR) results showed that the relative gene expression of ZO-1, occludin, MyD88, interleukin-1β (IL-1β), and IL-6 was decreased in the fluconazole-treated group compared to the control. Serum levels of IL-2, LZM, and IgM were significantly increased, while the IgG level was considerably downregulated in the fluconazole-treated compared to the control group. These results suggest that the administration of fluconazole can influence the gut microbiota and that a healthy gut microbiome is important for the regulation of the host immune responses.

INTRODUCTION

The gut microbiota is a complex and highly dynamic community of microorganisms that thrive in the gastrointestinal tract. It is now recognized as a promising contributing factor in determining the health and physiology of the host (1). The human microbiome has entered into the forefront of scientific research, with growing importance for both the medical and research communities, although current knowledge of the microbiome is scarce (2). Adverse changes in gut microbiota composition have been reported to be associated with a variety of human diseases, including inflammatory bowel disease (IBD), type 2 diabetes, atherosclerosis, and obesity disease (3–6). Thus, understanding alterations in the gut microbiota can play a role in the potential assessment of host health effects on disease initiation and progression.

Antibiotics that can treat or prevent infectious diseases play an important role in medical therapy (7). Fungi and bacteria encounter each other in various niches of the human body (8). They interact directly with one another or indirectly via the host response. Antibiotic usage has major impacts on the structure and function of the gut microbiota, often resulting in dysbiosis (9). Researchers used high-throughput sequencing techniques to explore the effects of antibacterial antibiotics on the composition of the human gut microbial community. They found that consumption of the antibiotic ciprofloxacin reduced the abundance, diversity, and uniformity of human gut microbiota; nevertheless, there was significant individual variation in the extent of such effects (10). Moreover, clinicians had found that antibiotic therapy caused exogenous vancomycin-resistant Enterococcus to almost completely replace the normal flora in the gut, disrupting the normal symbiotic gut flora (11). However, there are few reports of antifungal drugs affecting intestinal mucosal morphology, gut microbiota, and immunity. Fluconazole is a bis-triazole antifungal drug with novel pharmacokinetic properties (metabolic stability and relatively higher water solubility) that contribute to its therapeutic effects, and it is widely used to treat Candida infections (12). Thus, the aim of this study was to investigate the effects of fluconazole on intestinal microbiota (both bacteria and fungi) and immune response as well as the intestinal barrier in mice.

RESULTS

Gut microbiome composition.

We sequenced the 16S rRNA (bacteria) and internal transcribed sequence (ITS) region (fungi) genes to study the impact of fluconazole on the gut microbiome in mice. We found that the gut microbiome composition in mice changed significantly after fluconazole administration.

Bacteriome composition analysis.

As shown in Table 1, there were no differences in the bacterial species richness as measured by Chao1 (P = 0.9649, one-way analysis of variance [ANOVA]). However, Shannon's diversity in the fluconazole group was significantly reduced compared with the control group; global microbial composition was also found to be significantly different across the groups.

TABLE 1.

α-Diversity analysis of mouse gut microbiota in different group^a

Microbiota	Sample	Chao-1	Coverage	Shannon	Simpson
Bacteriome	Control	780.000	0.99	4.620	0.028
Fluconazole	766.330	0.99	4.242	0.050
Mycobiome	Control	270.500	0.99	3.828	0.072
Fluconazole	185.167	0.99	3.733	0.071

^aThere were no differences in the bacteria species richness as measured by Chao1 (P = 0.9649, one-way ANOVA) but significant differences in the fungi species richness (P = 0.0106, one-way ANOVA). However, Shannon's diversity of bacteria in the fluconazole group was significantly reduced compared with that of the control group. Shannon's diversity of fungi in the fluconazole group was not significantly different compared with the control group. Global microbial composition was also found to be significantly different across the groups.

At the phylum level, the gut microbiota of mice consists mainly of Actinobacteria, Bacteroidetes, Cyanobacteria, Deferribacteres, Epsilonbacteraeota, Firmicutes, Patescibacteria, Proteobacteria, and Tenericutes. The relative abundance of Bacteroidetes, Deferribacteres, Patescibacteria, and Tenericutes was significantly reduced after fluconazole treatment, and the relative abundance of Firmicutes and Proteobacteria was significantly increased (Fig. 1A and B). According to the analysis at the genus level, the relative abundances of Rikenella, Ruminiclostridium, Parvibacter, Parabacteroides, Oscillibacter, Mycobacterium, Mucispirillum, Faecalitalea, Escherichia Shigella, Candidatus Arthromitus, Alistipes, and Tyzzerella were significantly reduced in the fluconazole-treated group (Fig. 1C and D), and the principal-component analysis revealed no significant difference in bacterial composition between the two groups (Fig. 1E).

FIG 1.

Composition of murine fecal bacteriome with and without fluconazole treatment. (A and B) Analysis of bacterial species composition at the phylum level. (C and D) Analysis of bacterial species composition at the genus level. (E) β-Diversity analysis of mouse gut fungi in different groups. (F) The LDA scoring plot results of bacterial analysis.

The linear discriminant analysis (LDA) scoring plot results showed that the dominant bacteria in the control group were Ruminiclostridium_6, Aliihoeflea, Dubosiella, Turicibacter, Mucispirillum, Deferribacterales, Deferribacteraceae, Deferribacteres, Ruminococcaceae_UCG_013, Oscillibacter, Ruminiclostridium_9, Enterobacteriaceae, Enterobacteriales, Escherichia_Shigella, Erysipeloyrichia, Erysipeloyrichales, Erysipeloyrichaceae, and Gammaproteobacteria. The dominant bacteria in the fluconazole-treated group were Staphylococcus and Staphylococcaceae (Fig. 1F).

Mycobiome composition analysis.

As shown in Table 1, there were significant differences in the fungal species richness (P = 0.0106, one-way ANOVA). However, Shannon's diversity of fungi in the fluconazole group was not significantly different from that of the control group; global microbial composition was also found to be significantly different across the groups.

A total of 444 OTUs were common to both groups, 338 OTUs were unique to the control group, and 249 OTUs were unique to the fluconazole-treated group. Ascomycota and Basidiomycota were the dominant fungal flora of the mouse gut at the phylum level. The relative abundance of Ascomycota was significantly decreased and Mucoromycota was significantly increased in the intestine of mice in the fluconazole-treated group (Fig. 2A and B). Meanwhile, the relative abundances of Cladosporium and Fusarium were significantly higher in the fluconazole-treated group at the genus level, and the relative abundance of Mortierella was significantly lower. β-Diversity analysis revealed no significant differences in fungal floral composition between the control and fluconazole-treated groups (Fig. 2C).

FIG 2.

Composition of murine fecal mycobiome with and without fluconazole treatment. (A and B) Analysis of fungal species composition at the phylum level. (C) β-Diversity analysis of mouse gut fungi in different groups. (D) The LDA scoring plot results of fungal analysis.

The LDA score chart shows that the dominant fungi in the control group were Exophiala, Strophariaceae, Ophiocordyceps, and Ophiocordycipitaceae, and the dominant fungi in the fluconazole-treated group were Stachybotryaceae, Coprinellus, Leucosporidiaceae, Leucosporidiales, and Leucospordium (Fig. 2D).

Relative mRNA expression of cytokines and tight-junction (TJ) genes in the intestinal mucosa.

We investigated whether fluconazole treatment can influence the expression of immune-related genes in intestinal tissues. Reverse transcription-quantitative PCR (RT-qPCR) analysis of colon tissues showed that the relative gene expression of ZO-1, occludin, MyD88, interleukin-1β (IL-1β), and IL-6 was decreased in fluconazole-treated group mice compared to control group mice, while there was no significant difference in IL-8 levels, as shown in Fig. 3.

FIG 3.

Relative expression of cytokine and tight-junction genes in fluconazole-treated mice.

Serum cytokine levels.

To further investigate the systemic immune response to antibiotic treatment, we measured cytokine production at the protein level. Cytokine concentrations in serum were altered after treatment with antibiotics. Enzyme-linked immunosorbent assay (ELISA) results showed that serum levels of IL-2 (P < 0.05), LZM (P < 0.05), and IgM (P < 0.01) were significantly increased and IgG (P < 0.05) levels were significantly decreased after 7 days of fluconazole solution treatment in mice (Fig. 4).

FIG 4.

Effect of fluconazole administration on the level of immune-related molecules in the serum of mice.

Histopathological evaluations.

Intestinal histological evaluation of mice is summarized in Fig. S1 in the supplemental material. Colonic tissue specimen evaluation did not show any signs of mucosal layer disruption or increased infiltration of inflammatory cells in any groups.

DISCUSSION

The gastrointestinal tract is occupied by a diverse population of microorganisms, including bacteria, fungi, viruses, archaea, and protozoa. The gut microbiota plays various important roles in the health of the host, such as food digestion, production of nutrients, and stimulation of host immune response (8, 13). Antibiotics have been widely used for microbial infectious disease control, which may cause antibiotic-associated gut dysbiosis and lead to the development of diarrhea, mucosal immune disorders, and diarrheal disease (14). The antibiotic treatment causes the change of diversity and composition of gut microbiota in the host. Most of the research focused on the influence of antibacterial antibiotics on the bacteriome, and their studies demonstrated that different classes of antibiotic showed different effects on the gut microbiota (15). Since the fungal species comprise fewer than 1% of the microorganisms in the human intestinal tract, very little is known about the influence of antifungal drugs on gut microbiota (16). If gut bacterial and fungal communities inhabit the same habitats, they can undoubtedly influence each other (17). The major findings of our study indicated that the administration of antifungal drugs had relative effects on the composition of the microbiota (both bacteria and fungi) in mice compared to the control group. The results showed that after fluconazole administration, the gut microbial composition of mice was significantly changed. In the bacteriome, levels of Firmicutes and Proteobacteria significantly increased, while those of Bacteroidetes, Deferribacteres, Patescibacteria, and Tenericutes showed a remarkable reduction in the fluconazole-treated group compared to the control group. In the mycobiome, the relative abundance of Ascomycota was significantly decreased and Mucoromycota was significantly increased in the intestine of mice that were treated with fluconazole compared to the control group. Antibiotics have been shown to affect the gut microbiota in clinical trials. Some studies reported that oral treatment of mice with antifungal drugs resulted in increased disease severity in acute and chronic models of colitis, and microbiota profiling revealed the restructuring of fungal and bacterial communities. Specifically, representation of Candida spp. was reduced, while Aspergillus, Wallemia, and Epicoccum spp. were increased (18). In addition, fluconazole treatment was able to affect fungal translocation in patients with leaky gut (19). In the intestinal ecosystem, there are extensive interactions between fungi and bacteria, and fungi affect the composition of bacterial communities (20). In this experiment, the reason for the changes in intestinal microorganisms in mice after fluconazole administration is not only the direct effect of the drug but also the fungal-bacterial interactions.

Antibiotic treatment can not only disrupt intestinal physical barrier directly but also affect intestinal immune responses. Research has shown that some antibiotics can disrupt the gut physical barrier directly or indirectly (21, 22). We found that the administration of fluconazole showed no significant damage in histological level, but the expression of tight-junction genes decreased. Zonula occludens-1 (ZO-1) and occludin are two important TJ proteins that play critical roles in modulating intestinal epithelial barrier function (23–25). In this study, we found that the administration of fluconazole downregulated levels of ZO-1 and occludin, which is contrary to partial studies. Results from Shi et al. showed that the expression of the TJ proteins ZO-1 and occludin decreased about 50% in the colon after ampicillin treatment (22), while the Tulstrup et al. study showed that there are no significant differences in the expression level of ZO-1 and occludin between the control group and any of the antibiotic treatment groups (amoxicillin, cefotaxime, vancomycin, and metronidazole) (26). Gut microbiota dysbiosis can induce the alteration of immune responses and cause intestinal inflammation. Wheeler et al. demonstrated that fungal members of the gut community have surprisingly strong effects on the immunological responses of the host, dampening inflammatory responses in the gut despite their small numbers (20). In this study, we found that after fluconazole administration, the relative gene expression of IL-1β, IL-6, and IL-8 was decreased, serum levels of IL-2, LZM, and IgM were significantly increased, and IgG levels were significantly decreased in mice. These results indicated that fluconazole administration can increase the production of proinflammatory cytokines and stimulate innate immune responses in mice. Terada et al. showed that levels of inflammation-related cytokines (IL-1β, TGFb3, TGFb4, and IL-8) decreased after antibiotic treatment of chicks (27), but the results of Aguilera et al. showed that no differences in the expression of proinflammatory (IL-6, IL-12p40, and tumor necrosis factor alpha [TNF-α]) or anti-inflammatory (IL-10) cytokines in antibiotic-treated mice compared to the control group (28). In addition, Li et al. reported that antibiotic treatment increased the levels of IL-6, TNF-α, and IL-10 and decreased the levels of IL-17A, IL-22, IL-12, and gamma interferon (IFN-γ) in the blood (29). It has also been reported that fluconazole treatment in mice resulted in the elevation of serum Th2-associated antibodies, IgE, and HDM-specific IgG1 in response to HDM immunization as well as increases in Th2 cytokines (IL-4, IL-5, and IL-10). These findings provide compelling evidence to support a functional role of the fungal mycobiota and fungal-bacterial interactions in modulating immune function and development of inflammatory disease.

Theoretically, bacteria and fungi inhabiting the surface of intestinal mucosa or within the gut lumen may influence one another in response to the host and environmental factors. In this study, we investigated the antifungal administration and concomitant changes of gut bacteria and fungi in mice. Further, we have shown that the gut dysbiosis leads to immune imbalance. The results of this study suggested that gut bacterial-fungal interactions are vital considerations for antibiotics usage.

MATERIALS AND METHODS

Ethics statement.

The use of mice in this research was approved by the China Pharmaceutical University Animal Care and Use Committee, and all animal experimental procedures were in accordance with the guidelines of the Institute Animal Care and Use Committee of China Pharmaceutical University.

Animal and antibiotic treatment.

Female mice (n = 20), obtained from the Model Animal Research Center of Nanjing University (Nanjing, China), were kept in cages with free access to commercial food and water. A total of 20 mice were randomly divided into control and antibiotic treatment groups after 2 weeks of acclimatization feeding. The antibiotic treatment group was administered, by oral gavage, 0.3 ml fluconazole (2 mg/ml) every day, and the control group was gavaged with equal volumes of 0.9% NaCl. After 1 week of treatment, all mice were sacrificed. Blood, colon contents, and colon tissues were collected for further analysis after the sacrificing.

Gut microbiome analysis.

The V3–V4 region of the gut bacterial 16S rRNA and fungal ITS genes was amplified using universal primer 341F/806R (338F, 5′-CCTAYGGGRBGCASCAG-3′; 806R, 5′-GGACTACNNGGGTATCTAAT-3′) and ITS1F/ITS2R (ITS1F, 5′-CTTGGTCATTTAGAGGAAGTAA-3′; ITS2R, 5′-GCTGCGTTCTTCATCGATGC-3′) by PCR. The amplified products were then purified by a TIANGEN DNA gel purification kit (TIANGEN Mini purification kit; Beijing, China). MiSeq library construction and sequencing were performed using the Illumina MiSeq PE300 platform (Illumina, San Diego, CA, USA). The reads were filtered by QIIME (Quantitative Insights Into Microbial Ecology) quality filters. All the bioinformatics data were analyzed on the free online platform of Majorbio Cloud Platform (www.majorbio.com).

RNA isolation and RT-qPCR of proinflammatory cytokines and tight-junction genes.

After 1 week of treatment, colonic tissues were collected and quickly frozen in liquid nitrogen. Samples were kept at −80°C until RNA extraction. Total RNA from the intestinal tissue was extracted using TRIzol reagent (Invitrogen, USA). Quantitative real-time PCR used a one-step TB green PrimeScript RT-PCR kit and was performed in a StepOne Plus real-time PCR system (Applied Biosystems), as previously described. Relative gene expression was calculated using the comparative threshold cycle method (2^−△△CT). The primer sequences are shown in Table S1 in the supplemental material.

ELISA.

Enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of IL-2, lysozyme (LZM), IgM, and IgG in the serum of mice. In brief, blood from mice was harvested by removing the eyeballs after centrifugation at 3,000 rpm for 10 min. ELISA analysis was performed using 10 μl of serum according to the manufacturer's protocol (YingxinLab, Shanghai, China).

Histopathological examination.

Colonic tissue samples were fixed in a 10% buffered formalin solution for histopathology. Tissue samples were washed with tap water before routine serial treatment of samples with graded alcohol and xylene, performed in the Shandon Citadel 2000 tissue system. After routine histopathological processing, all samples were embedded in a paraffin block and 5-mm sections were prepared using a rotary microtome (Leicia RM 2255; Wetzlar, Germany). All sections were stained with hematoxylin and eosin (H&E) for standard histopathological evaluation. Slides were taken by using an optomicroscope (Olympus BX51 with DP72 camera attachment; Tokyo, Japan).

Data availability.

All sequence data have been deposited in the NCBI database (accession number SRP265194).