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. 2021 Jan 20;6(1):e00982-20.
doi: 10.1128/mSphere.00982-20.

Decreased Ecological Resistance of the Gut Microbiota in Response to Clindamycin Challenge in Mice Colonized with the Fungus Candida albicans

Laura Markey  1   2 , Antonia Pugliese  1   2 , Theresa Tian  3 , Farrah Roy  4 , Kyongbum Lee  3 , Carol A Kumamoto  5
Affiliations

Decreased Ecological Resistance of the Gut Microbiota in Response to Clindamycin Challenge in Mice Colonized with the Fungus Candida albicans

Laura Markey et al. mSphere. .

Abstract

The mammalian gut microbiota is a complex community of microorganisms which typically exhibits remarkable stability. As the gut microbiota has been shown to affect many aspects of host health, the molecular keys to developing and maintaining a "healthy" gut microbiota are highly sought after. Yet, the qualities that define a microbiota as healthy remain elusive. We used the ability to resist change in response to antibiotic disruption, a quality we refer to as ecological resistance, as a metric for the health of the bacterial microbiota. Using a mouse model, we found that colonization with the commensal fungus Candida albicans decreased the ecological resistance of the bacterial microbiota in response to the antibiotic clindamycin such that increased microbiota disruption was observed in C. albicans-colonized mice compared to that in uncolonized mice. C. albicans colonization resulted in decreased alpha diversity and small changes in abundance of bacterial genera prior to clindamycin challenge. Strikingly, co-occurrence network analysis demonstrated that C. albicans colonization resulted in sweeping changes to the co-occurrence network structure, including decreased modularity and centrality and increased density. Thus, C. albicans colonization resulted in changes to the bacterial microbiota community and reduced its ecological resistance.IMPORTANCECandida albicans is the most common fungal member of the human gut microbiota, yet its ability to interact with and affect the bacterial gut microbiota is largely uncharacterized. Previous reports showed limited changes in microbiota composition as defined by bacterial species abundance as a consequence of C. albicans colonization. We also observed only a few bacterial genera that were significantly altered in abundance in C. albicans-colonized mice; however, C. albicans colonization significantly changed the structure of the bacterial microbiota co-occurrence network. Additionally, C. albicans colonization changed the response of the bacterial microbiota ecosystem to a clinically relevant perturbation, challenge with the antibiotic clindamycin.

Keywords: Candida albicans; ecological resistance; microbiota.

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Figures

FIG 1
FIG 1
Effects of C. albicans colonization on the composition of the bacterial microbiota. Mice were pretreated with cefoperazone in drinking water for 10 days and then either colonized with C. albicans (5 × 107 CFU by oral inoculation) or not. Mice were then switched back to sterile water, and the microbiota were allowed to recover for 3 weeks. (A) Experimental design timeline. Fecal pellets were collected (black arrows with numbers indicating day of collection) for bacterial microbiota analysis by 16S rRNA sequencing. Mice were from two separate experimental trials; total uncolonized, N = 20, and C. albicans colonized, N = 24. (B) qPCR of universal 16S rRNA sequence was used to measure the total bacterial DNA content (in arbitrary units) per gram of feces for each sample used for sequencing analysis. (C) Relative abundance of genera in the pre-clindamycin (day −1) microbiota of uncolonized mice and C. albicans-colonized mice. Genera shown were identified as differential and significantly associated with C. albicans colonization status by LEfSe analysis. Bars indicate the averages and error bars the standard errors of the means (SEMs). Corio, unclassified Coriobacteriaceae; Anaero, Anaeroplasma; Bact, Bacteroides; Turic, Turicibacter; Copro, Coprococcus; Clost, Clostridiaceae Clostridium. (D) Alpha diversity of the pre-clindamycin microbiota of uncolonized mice and mice colonized with C. albicans as measured by the Simpson index. Symbols indicate the diversity indices of individual mouse microbiota and bars indicate the averages. (E) Principal-coordinate analysis of the weighted UniFrac distance matrix of the pre-clindamycin bacterial microbiota of uncolonized mice (●) and mice colonized with C. albicans (△).
FIG 2
FIG 2
C. albicans colonization altered network topology of the bacterial microbiota. Mice were pretreated with cefoperazone in drinking water for 10 days and then either colonized with C. albicans (5 × 107 CFU by oral inoculation) or not. Mice were then switched back to sterile water, and the microbiota were allowed to recover for 3 weeks. 16S rRNA sequencing was used to analyze the bacterial microbiota of fecal pellets collected prior to clindamycin treatment (day −1). The sequencing data were analyzed using QIIME2 and taxonomic identification of amplicon sequence variants (ASVs) at the genus level performed by matching to the Greengenes database. Absolute abundance of each genus was determined by multiplying the relative abundance of that genus by the total quantity of bacterial DNA normalized to grams of fecal pellet material measured by qPCR using universal primers. The absolute abundance of each genus was correlated with the abundance of every other genus using Pearson correlation, and this correlation matrix was used for network analysis (A to D) using qgraph. Each node represents a genus (described in Table 1), and each edge and its thickness represent the strength (R) of the correlation between those nodes. Only statistically significant edges are shown, P < 0.05 after Benjamini-Hochberg correction. Positive correlations are shown as black edges and negative correlations are red edges. (A and B) Co-occurrence network of the uncolonized bacterial microbiota. (C and D) C. albicans-colonized microbiota network. Nodes are colored by cluster (A and C) as determined by fast-greedy cluster analysis using igraph, such that different colors indicate separate community clusters, and by taxonomic class (B and D), for which the legend is shown far right. Network analysis of the pre-clindamycin microbiota included mice from two cohorts: total uncolonized, N = 20; C. albicans colonized, N = 24.
FIG 3
FIG 3
C. albicans colonization decreased ecological resistance of the bacterial microbiota. Mice were pretreated with cefoperazone in drinking water for 10 days and then either colonized with C. albicans (5 × 107 CFU by oral inoculation) or not. Mice were then switched back to sterile water, and the microbiota were allowed to recover for 3 weeks. They then received a single intraperitoneal (i.p.) injection of 1.11 mg/kg body weight, 3.33 mg/kg, or 10 mg/kg clindamycin. Fecal pellets were collected 1 day before and 1 day after clindamycin treatment for bacterial microbiota analysis by 16S rRNA sequencing and QIIME2. (A) Relative abundances of all bacterial genera detected with a median of >0 pre-clindamycin, before and after clindamycin challenge, in the average uncolonized and C. albicans-colonized microbiota. The colored proportion of the bar represents the average proportion of the whole microbiota for each individual genus. The most abundant bacterial genera are shown in the legend at the right: Bact, Bacteroides; Clostr. 2, unclassified Clostridiales 2; Akk, Akkermansia; S24-7, unclassified S24-7; Lachno, unclassified Lachnospiraceae; Allo, Allobaculum; Oscil, Oscillospira; Lachno. 2, unclassified Lachnospiraceae 2; Rumin, unclassified Ruminococcaceae; Lacto, Lactobacillus; Bifido, Bifidobacteria; Sutt, Sutterella. (B) Simpson indices before and after clindamycin challenge in uncolonized and C. albicans-colonized mice. Symbols indicate individual mouse microbiota, bars indicate the averages, and error bars indicate SEMs. Gray bars/● indicate uncolonized mice and open bars/△ C. albicans colonized. ANOVA followed by Sidak’s post hoc test: **, P < 0.01; ****, P < 0.0001. Average changes in weighted UniFrac position from starting position of the uncolonized microbiota (C) and the C. albicans-colonized microbiota (D) as a consequence of clindamycin treatment, change in position calculated for each individual, and the average of experimental groups. Symbols indicate the averages and error bars indicate the SEMs. Welch’s t test was used to compare experimental groups; except where noted, P < 0.05. Uncolonized (C): 1.11 mg/kg, N = 6; 3.33 mg/kg, N = 7; 10 mg/kg, N = 7. C. albicans-colonized (D): 1.11 mg/kg, N = 8; 3.33 mg/kg, N = 8; 10 mg/kg, N = 8.
FIG 4
FIG 4
Clindamycin challenge has a differential effect on uncolonized and C. albicans-colonized microbiota networks. Network analysis was performed as described for Fig. 2. Only the pre-clindamycin network is shown, with nodes colored according to post-clindamycin response. (A to C) Co-occurrence network of the uncolonized bacterial microbiota. (D to F) C. albicans-colonized microbiota network. Nodes are colored by their response in fold change in absolute abundance after challenge with low-dose clindamycin (1.11 mg/kg), intermediate-dose clindamycin (3.33 mg/kg), or high-dose clindamycin (10 mg/kg). The legend is shown at the bottom right. Uncolonized (N = 20 total): 1.11 mg/kg, N = 6; 3.33 mg/kg, N = 7; 10 mg/kg, N = 7. C. albicans colonized (N = 24 total): 1.11 mg/kg, N = 8; 3.33 mg/kg, N = 8; 10 mg/kg, N = 8.
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