Establishment and characterization of stable, diverse, fecal-derived in vitro microbial communities that model the intestinal microbiota

Abstract

Efforts to probe the role of the gut microbiota in disease would benefit from a system in which patient-derived bacterial communities can be studied at scale. We addressed this by validating a strategy to propagate phylogenetically complex, diverse, stable, and highly reproducible stool-derived communities in vitro. We generated hundreds of in vitro communities cultured from diverse stool samples in various media; certain media generally preserved inoculum composition, and inocula from different subjects yielded source-specific community compositions. Upon colonization of germ-free mice, community composition was maintained, and the host proteome resembled the host from which the community was derived. Treatment with ciprofloxacin in vivo increased susceptibility to Salmonella invasion in vitro, and the in vitro response to ciprofloxacin was predictive of compositional changes observed in vivo, including the resilience and sensitivity of each Bacteroides species. These findings demonstrate that stool-derived in vitro communities can serve as a powerful system for microbiota research.

Keywords: antibiotics; ciprofloxacin; culturomics; ecological stability; ex vivo; gut microbiota; microbial ecology; microbiota perturbations; synthetic communities.

Figure 1:. High-throughput cultivation of feces from humanized mice yields stable, complex, and reproducible microbial communities.

A) Experimental setup. germ-free mice were colonized with feces from a single human donor (“humanized”). Fecal samples from humanized mice were inoculated into anaerobic batch culture and passaged with dilution every 48 h to derive SICs. B) In vitro passaging produces stable and complex SICs. Family-level compositions of a representative SIC derived from the feces of MD mouse 1 during 16 rounds of in vitro passaging in BHI. C) In vitro passaging can produce an SIC that resembles the fecal inoculum. Family-level relative abundances (mean of passages 4–16) for the SIC in (B) compared with the fecal inoculum from which it originated. D) In vitro-passaged SICs are highly reproducible. ASV-level relative abundances for two technical replicates of the SIC in (B) after 7 passages. R and p were computed using only ASVs present in both samples. Relative abundances <10⁻⁴ in (C,D) were set to 10⁻⁴ for visualization.

Figure 2:. Re-introduction of SICs into a host re-establishes gut microbiota composition and promotes host homeostasis.

A) An SIC generated from a humanized mouse was used to colonize germ-free mice to determine whether SICs can re-establish the original microbiota. B) SIC colonization of germ-free mice restored families overrepresented in vitro back to levels similar to those in the humanized mice from which the SIC was derived. Comparison of the family-level relative abundances of a humanized-mouse fecal inoculum and the mean family-level relative abundance of germ-free mice colonized with an SIC derived from the humanized-mouse fecal inoculum (inoculum n=1, SIC-colonized n=3). Relative abundances <10⁻⁴ were set to 10⁻⁴ for visualization. C-E) C-E) The host proteomes in humanized and SIC-colonized mice were more highly correlated (C) than either set of mice with germ-free mice (D and E). Immune-related proteins (red) were similarly up-regulated in humanized and SIC-colonized mice relative to germ-free mice.

Figure 3:. High-throughput cultivation of humanized-mouse feces preserves inoculum composition and richness.

A) Experimental setup. Germ-free mice colonized with feces from a single human donor (“humanized”) were fed an SD or an MD and treated with ciprofloxacin for 5 days. Fecal samples from two mice on each diet were collected on four days (0, 1, 5, 14) before, during, at the end of, and after treatment, and were inoculated into anaerobic batch culture and passaged with dilution every 48 h to derive SICs. Sixteen samples (2 diets, 2 mice, 4 time points during ciprofloxacin treatment) were inoculated into 4 media (BHI, TYG, GAM, and YCFA) in triplicate. B) Diet alters the trajectory of microbiota reorganization during ciprofloxacin treatment in vivo. PCoA of community composition of the fecal inocula using unweighted Unifrac distance computed on all in vivo and in vitro samples at the ASV level. Lines (black and grey) correspond to two different mice. C) Medium and inoculum determine the final composition of passaged SICs. The 7^th passage of all 192 SICs is shown in a PCoA of SIC composition using unweighted Unifrac distance computed on all in vivo and in vitro samples at the ASV level. Left: samples are colored by media, with shapes representing the diet in the mice from which the inocula were taken. Symbols are the centroid of three replicates, with lines connecting the replicates to the centroid. Right: SICs derived in BHI with colors and shapes representing the timepoint during ciprofloxacin treatment and diet, respectively, in the mice from which the inocula were taken. Symbols are the centroid of three replicates, with lines connecting the replicates to the centroid. Original fecal inocula are plotted in light colors. D) Most steady-state SICs are similar to the fecal samples from which they were derived, as shown by weighted Unifrac distance of the 7^th passage of each SIC to the corresponding fecal inoculum. Colored circles, mean distance for each medium; individual SICs in gray. Error bars, standard deviations; n=48. Dashed line, mean distance between fecal samples. E) SIC diversity correlates with inoculum diversity in BHI. Richness (number of ASVs in rarefied data) was compared for the 7^th passage of each SIC and the corresponding fecal inocula, and separated and colored/shaped as in (C). R and p are for Pearson coefficient; n=48.

Figure 4:. Pre-exposure of the gut microbiota to ciprofloxacin in vivo results in differential invasion of S. Typhimurium in vitro.

A) Experimental setup for in vitro challenge with S. Typhimurium. SICs passaged in BHI from pre-, peak, residual, and post-treatment humanized mouse fecal inocula were revived after freezing and passaged twice in BHI. SICs were mixed with S. Typhimurium and S. Typhimurium levels were quantified after 48 h of growth. B) SICs derived from mice treated with ciprofloxacin are more susceptible to S. Typhimurium. Colonies of S. Typhimurium SL1344 after 48 h of growth with SICs diluted 1:10⁴ and grown aerobically on LB+streptomycin. C) Single-cell quantification of mCherry-tagged S. Typhimurium 14028s after co-culture with SICs derived from pre- and residual-treatment mice fecal inocula. p-value is from a Student’s two-sided t-test; n=3.

Figure 5:. In vitro treatment of an SIC with ciprofloxacin results in changes in community composition consistent with strain sensitivity.

A) Changes in family-level abundances during ciprofloxacin treatment in vivo can be predicted by strain-level sensitivities in vitro. Fractional changes were computed as the ratio of residual and pre-treatment time-point abundances in vivo. MIC is the mean value across strains in a given family determined from growth in isolation in BHI (Table S1). B) Experimental setup. An SIC passaged in BHI from a pre-treatment humanized mouse fecal inoculum (Pre-SD) was passaged in ciprofloxacin three times (i,ii,iii). C) Pre-SD SIC yield and growth rate decreased with increasing concentrations of ciprofloxacin. OD was measured after 48 h of growth with ciprofloxacin. Lines, means of triplicate growth curves; error bars, standard deviations. D) Richness of the Pre-SD SIC decreased in a dose-dependent manner. Data are means of two technical replicates. E) Ciprofloxacin treatment in vitro selects for a few families. Family-level composition of the Pre-SD SIC across passages in ciprofloxacin. The limit of detection was ~10⁻³. Data are means of two technical replicates. F) Growth of ASVs in the Pre-SD SIC under ciprofloxacin treatment can be predicted by their sensitivity in isolation. Fractional-abundance changes of ASVs corresponding to the 15 isolates in (A) were computed after one passage in 2 μg/mL ciprofloxacin.

Figure 6:. In vitro treatment of an SIC with ciprofloxacin results in similar responses as in vivo.

A) Experimental setup mimicking transient treatment in vivo. An SIC passaged in BHI from a pre-treatment humanized mouse fecal inoculum (Pre-SD) was passaged in ciprofloxacin three times (i,ii,iii); or in ciprofloxacin once and then twice without the drug (i,iv,v). B) Richness of the Pre-SD SIC recovered only partially after removal of ciprofloxacin (green) versus continuous exposure (blue). Data are means of two technical replicates. C) Some families (e.g. Erysipelotrichaceae and Ruminococcaceae) recover after transient ciprofloxacin treatment. Data are the mean log₁₀(relative abundance) at the family level of two replicates during one round of ciprofloxacin treatment and two rounds of recovery. D) Ciprofloxacin-induced changes to family-level abundances in SD mice in vivo resemble changes in vitro in (C). Data are the mean log₁₀(relative abundance) at the family level of two mice. E) Most families display similar qualitative abundance changes in vivo and in vitro.

Figure 7:. In vitro treatment of an SIC reveals resistance and resilience in the Bacteroides genus.

A) Bacteroides dynamics in vivo consist of B. vulgatus dominance during treatment and the recovery of several species after treatment. Data are the mean log₁₀(relative abundance) at the ASV level across two SD mice: Bacteroides ovatus (Bo), B. vulgatus (Bv), B. uniformis (Bu), B. caccae (Bc), B. intestinalis (Bi), B. fragilis (Bf), and B. thetaiotaomicron (Bt). Other Bacteroides are shown in shades of grey. B,C) Bacteroides survival and recovery in vivo can be respectively explained by resistance and resilience characteristics of the Pre-SD SIC in vitro during continuous treatment (B) or one round of treatment followed by two rounds of recovery (C). Data are the mean log₁₀(relative abundance) of two replicates during one round of ciprofloxacin treatment and two rounds of recovery at the ASV level. Thick lines highlight Bacteroides ASVs that show resistance (B) or resilience (C) at the highest concentration that they display the behavior.