Perturbations in the gut microbiome of C57BL/6 mice by the sobriety aid Antabuse^® (disulfiram)

Abstract

Aims

Disulfiram (Antabuse^®) is an oral alcohol sobriety medication that exhibits antimicrobial activity against Gram-positive facultative anaerobes. The aims of this study were to measure the antimicrobial activity against anaerobic bacteria of the gut human microbiome and establish the extent that disulfiram alters the microbial composition of the ileum, cecum, and feces using C57BL/6 mice.

Methods and results

Antimicrobial susceptibility testing by the microdilution method revealed that disulfiram inhibits the in vitro growth of gut anaerobic species of Bacteroides, Clostridium, Peptostreptococcus, and Porphyromonas. Differential sequencing of 16S rRNA isolated from the ileum, cecum, and feces contents of treated vs. untreated mice showed that disulfiram enriches the Gram-negative enteric population. In female mice, the enrichment was greatest in the ileum, whereas the feces composition in male mice was the most heavily altered.

Conclusions

Daily administration of oral disulfiram depletes the enteric Gram-positive anaerobe population as predicted by the minimum inhibitory concentration data for isolates from the human gut microbiota.

Impact Statement.

The study provides evidence that prolonged use of disulfiram can result in gastrointestinal disturbances precipitated by depletion of beneficial anaerobes. When this occurs, probiotics may aid in restoring gut health or switching to an alternative therapy to support alcohol abstinence.

Introduction

Disulfiram (DSF) is an aversion-based therapy for the treatment of alcohol use disorder (Kranzler 2023). Marketed under the brand name Antabuse^®, DSF tablets are taken in 125, 250, or 500 mg daily regimens over a period of months to years to foster alcohol abstinence (Reus et al. 2018). Upon ingestion, DSF is partially metabolized to N,N-diethyldithiocarbamate (DDTC) in the gastrointestinal tract and absorbed into circulation to enact the inhibitory effects on ethanol metabolism (Eneanya et al. 1981). First-pass metabolism by the hepatic cytochrome P450 system further transforms the DDTC into tertiary metabolites that work in concert to inhibit liver detoxification of ethanol-derived acetaldehyde. The resultant acetaldehyde accumulation in tissues induces severe nausea and headache to deter alcohol consumption.

Although regulatory approval is limited to use as a sobriety aid, additional indications are being sought to repurpose DSF in medicine. Clinical trials have evaluated DSF in therapies for cocaine and methamphetamine dependence (NCT00395850, NCT00731133), recurrent glioblastoma (NCT02678975), HIV latency (NCT01286259), and post-treatment Lyme disease syndrome (NCT03891667). Moreover, extensive efforts have been made to repurpose DSF as an antimicrobial drug due to its diverse activity spectrum encompassing bacteria, yeasts, molds, viruses, protozoa, and helminths (Custodio et al. 2022). A recent study reported that the antibacterial activity spectrum of DSF and its metabolites is mostly confined to Gram-positive species with Staphylococcus and Bacillus species exhibiting the highest degree of susceptibility based on minimum inhibitory concentration (MIC) measurements (Frazier et al. 2019). The antimicrobial spectrum of DSF and DDTC further extends to fungi of the human microbiome, including Candida albicans (Shanholtzer et al. 2022).

Due to its antimicrobial properties and prolonged oral use (Custodio et al. 2022), Antabuse^® has the potential to elicit perturbations in the gut microbiota. The intestinal microbiome, consisting principally of obligate anaerobes (e.g. Bacteroides, Clostridium, and Lactobacillus) and Enterobacteriaceae, has beneficial effects on the immune system (Zheng et al. 2020), colonization resistance (Ducarmon et al. 2019), and mental (Clapp et al. 2017), dermal (Byrd et al. 2018), and cardiovascular (Wang et al. 2022) health. With extensive evidence indicating that an altered microbiome can precipitate or exacerbate human disease (Kho et al. 2018), there is a need to establish the impact of DSF on the gut composition.

The following report describes the extent to which a short-course, high-dose regimen of oral DSF modifies the gut microbiome of C57/B6 mice. Two recent articles gave a partial account of the influence by analyzing the fecal composition in male murine models of nonalcoholic steatohepatitis (Lei et al. 2022) and atherosclerosis (Traughber et al. 2024). Because the in vivo studies were conducted with disease models and applied other variables, the current work sought to establish the impact of DSF on the intestinal microbiota of wild-type C57BL/6 mice. The ileum, cecum, and feces contents were harvested from both female and male mice treated for 1 week with either DSF or vehicle control for differential and comparative analysis of the gut microbiomes. We further evaluated a panel of anaerobic species from the human microbiome against DSF and DDTC to establish whether the MIC data are translated in vivo.

Materials and methods

Culture methods

As part of the Human Microbiome Project, bacteria strains from BEI Resources (NIH/NIAID) were cultured from frozen stocks stored at −80°C. The isolates were grown at 37°C in an AS-580 anaerobic chamber (Anaerobe Systems, CA, USA) and subcultured twice prior to testing. Lactobacillus species were enumerated in deMan, Rogosa, and Sharpe broth (MRS, Difco). All other anaerobic species were grown on CDC anaerobic blood agar (Anaerobe Systems) and then in brain heart infusion broth (BHI, Difco) or reinforced clostridial medium (RCM, Difco).

Antimicrobial testing

MICs were determined by the microdilution assay in accordance with CLSI M11-A7 protocols for antimicrobial testing of anaerobic bacteria. Drug stocks of 0.5 mg ml⁻¹ DSF (≥97.0%, TCI America) were prepared daily in dimethyl sulfoxide (DMSO) and evaluated at a maximal concentration of 16 µg ml⁻¹ due to low water solubility. All other drugs were prepared as 1 mg ml⁻¹ stocks (water) and tested at a concentration in the range of 0.25–32 µg ml⁻¹. Overnight broth cultures were standardized to a 0.5 McFarland saline suspension and diluted to 10⁵ CFU ml⁻¹ in broth. Due to rapid decomposition of DSF from the high cysteine content of RCM, antimicrobial testing was performed in BHI broth. Flat-bottom 96-well microtiter plates containing 2-fold serial dilutions of drug were inoculated and incubated in an AS-580 anaerobic chamber with an atmosphere of 85% N₂, 10% CO₂, and 5% H₂. The observed MIC endpoints were recorded for treatments that conferred complete visual growth inhibition at 24 hours or until growth was detected in untreated wells.

Animal ethics

Animal studies were conducted in an AAALAC-accredited animal care facility on the Marshall University campus (A3578-01). Animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) prior to initiation. All protocols were performed in accordance with the guidelines and regulations in the Guide for the Care and Use of Laboratory Animals: Eighth Edition.

Animal protocol

Inbred 6-week-old male (20–25 g) and female (17–20 g) C57BL/6 mice were obtained from Hilltop Lab Animals, Inc. (Scottdale, PA, USA) and housed four per cage. The animals were maintained under a controlled temperature (21–23°C) with a 12-hour light/dark cycle and provided irradiated pelleted food and water ad libitum. The mice were acclimated for a minimum of 7 days to their surroundings. The animals trained to accept oral delivery by a pipette tip of the drug vehicle consisting of autoclaved sweetened condensed milk (Nestlé^® Carnation^®) diluted by 50% with sterile water (Scarborough et al. 2020). Female (n = 16) and male (n = 16) mice were randomly assigned into two groups to receive either vehicle or DSF (α = 0.05, power = 0.8). A total of 8 female and 8 male mice per treatment group were used to assess for statistical significance at P < 0.05. On day 1, the drug-treated group received two 100 mg kg⁻¹ doses per day from a 20 mg ml⁻¹ DSF stock suspension separated by at least 8 hours. The control group received equal volumes of vehicle without DSF based on body weight. On day 7, fecal pellets and the contents of the ileum and cecum were collected. The intestines were sectioned and flushed with sterile phosphate buffer saline (pH 7.4). The mucosal linings were scraped with a glass microscope slide to isolate the small and large intestines microflora for next-generation sequencing (NGS).

16S rRNA sequencing

The samples were processed using the Qiagen QIAamp^® PowerFecal^® Pro DNA Kit (Germantown, MD, USA) according to the manufacturer’s instructions. The genomic material was then submitted to CD Genomics (Shirley, NY, USA) for processing through their workflow consisting of PCR amplification, quantification, purification, DNA library construction, and sequencing. The V4 hypervariable region of the bacterial 16S rRNA gene was used in amplification. Amplicons were sequenced on a paired-end Illumina MiSeq™ platform (Illumina, CA, USA) using the PE250 sequencing strategy to generate 300 bp paired-end raw reads, and then pretreated. Paired-end reads were merged using Flash (v1.2.1) (Magoč et al. 2011) and quality filtering was performed on raw tags under the fastp quality control process. Quantitative Insights in Microbial Ecology (QIIME 2) (Bolyen et al. 2019) was used to cluster the tags and acquire the operational taxonomic units (OTUs). Taxonomic assignments were performed using a naive Bayes classifier that was trained on the SILVA 138 99% OTUs (Quast et al. 2013).

Bioinformatics analysis of amplicon sequence data

All analyses were performed in R (R Core Team) (version 1.34.0). The R package phyloseq (McMurdie et al. 2013) was used to remove taxa (R Core Team) with <20 total OTU counts across all samples (phyloseq::prune_taxa) and samples with total OTU counts <2000 (phyloseq::prune_samples). The filtered count table was normalized by dividing each sample OTU count by the total counts for that sample, resulting in sample counts of each sample adding to 1. Dimensionality reduction analysis was performed using nonmetric multidimensional scaling (NMDS) with a Bray–Curtis distance method (phyloseq::ordinate, method = “NMDS,” distance = “bray”) on normalized OTU counts. Differential expression (DE) analysis was performed via the R package DESeq2 (Love et al. 2014) on nonnormalized OTU counts at the species, genus, and family taxonomic levels by combining OTUs of the same taxonomic classification (phyloseq::tax_glom, naRM = T). Comparisons used a Wald significance test and a parametric fit of dispersion to mean values (DESeq2::DESeq, test = “Wald,” fittype = “parametric”) with 0 OTU counts being adjusted (DESeq2::estimateSizeFactors, type = “poscounts”). Nonnormalized OTU counts combined at the species, genus, and family levels (phyloseq::tax_glom, NArm = T) were estimated for alpha diversity richness metrics [phyloseq::estimate_richness, measures = (“Observed,” “Chao1,” “ACE,” “Shannon,” “Simpson,” “InvSimpson,” “Fisher”)]. Each richness metric was compared between treatments using a Wilcoxon rank-sum test and adjusted for multiple testing using the Bonferroni–Holm method (stats::pairwise.wilcox.test, p.adjust.method = “holm”) at a significance threshold of 0.05. All other statistical analyses were performed in either Microsoft Excel or GraphPad Prism v10.2.3. Normalized gene expression data were log transformed and presented as fold change (FC) values with their P-values with statistical significance defined at P < 0.05.

Results

Antimicrobial testing

Table 1 depicts the MIC values of DSF and DDTC for authenticated anaerobic strains isolated as part of the Human Microbiome Project. The table shows that DSF inhibited the growth of seven Gram-positive and Gram-negative species at 16 µg ml⁻¹ or less. Bacteria exhibiting the highest susceptibility were Peptostreptococcus and Porphyromonas, while commensal species of Bifidobacterium, Lactobacillus, and Ruminococcaceae displayed MICs over 16 µg ml⁻¹. Other core gut microbiota species of Bacteroides and Clostridium demonstrated weak overall susceptibility to DSF. Similarly, anaerobes with susceptibility to DSF were inhibited by DDTC but at 2- to 4-fold higher concentrations. Lastly, the MIC data indicate that metronidazole and vancomycin are more potent inhibitors of anaerobic bacteria growth than either DSF or DDTC.

Table 1.

In vitro susceptibility of microflora anaerobes to DSF and metabolite DDTC.

			MIC (µg ml−1)a
Species	Strain	Source	DSF	DDTC	MET	VAN	COL
Anaerococcus lactolyticus	CC31C	Colon	>16	>32	1	0.5	–
Bacteroides eggerthii	1_2_48 FAA	Intestines	8	16	≤0.25	–	>32
Bacteroides fragilis	CL07T12C05	Feces	16	>32	≤0.25	–	>32
Bifidobacterium breve	JCP7499	Vagina	>16	>32	>32	0.5	–
Bifidobacterium longum	CMW7750	Vagina	>16	>32	4	0.5	–
Clostridiales bacterium	3_1_39B/D5	Intestines	16	>32	≤0.25	1	–
Clostridium innocuum	6_1_30	Colon	>16	>32	≤0.25	16	–
Clostridium symbiosum	WAL-14 673	Feces	16	>32	≤0.25	0.5	–
Eubacterium infirmum	F0142	Dental plaque	16	16	≤0.25	1	–
Fusobacterium nucleatum	MJR7757B	Vagina	>16	>32	≤0.25	≤0.25	–
Lachnospiraceae sp.	6_1_63FAA	Colon	>16	>32	≤0.25	0.5	–
Lactobacillus paragasseri	JV-V03	Vagina	>16	>32	>32	2	–
Lactobacillus jensenii	EX849587VC06	Vagina	>16	>32	>32	4	–
Lactobacillus johnsonii	135–1-CHN	Vagina	>16	>32	>32	2	–
Parabacteroides distasonis	31_2	Feces	>16	>32	≤0.25	–	>32
Peptostreptococcus micros	CC57A	Colon	8	>32	≤0.25	0.5	–
Peptostreptococcus sp.	MV1	Vagina	2	4	≤0.25	≤0.25	–
Porphyromonas uenonis	UPII 60–3	Vagina	0.5	2	≤0.25	–	>32
Prevotella bivia	GED7880	Vagina	>16	>32	1	–	8
Ruminococcaceae sp.	D16	Colon	>16	>32	≤0.25	8	–

^aDSF—disulfiram; DDTC—sodium diethyldithiocarbamate; VAN—vancomycin HCl; MET—metronidazole; COL—colistin sulfate.

Effects on alpha diversity

Alpha diversity is a holistic method to assess taxa richness and evenness in a defined environment. Indices such as Chao1 and Shannon are often employed in comparative analyses of alpha diversity between microbiome habitats. Chao1 estimates taxa richness by accounting for OTUs, species, or genera, while Shannon diversity measures are based on taxa richness and abundance or evenness (Willis 2019). Figure 1 compares the alpha diversity in the ileum, cecum, and feces of untreated (null) vs. treated mice. The Chao1 results showed no statistical differences in alpha diversity at the species level (P > 0.05). However, DSF induced increases in alpha diversity at the family and genus levels for male fecal microbiomes (P 0.01) (Supplementary Table 1S). No statistical differences were likewise observed in the Shannon indices at neither the species nor other taxa levels. Moreover, NMDS plots showed little segregation for treated and untreated microbiomes within the same sex (Supplementary Fig. 1S). Intergender comparisons of alpha diversity revealed statistical similarities between ileum and cecum microbiota, but greater diversity was present in male feces regardless of treatment.

Figure 1.

Comparison of microbial richness (alpha diversity) stratified by Chao1 and Shannon index values at the species level for ileum, cecum, and feces. Each dot represents the richness value for one animal and the boxplots depict the median range of the cohort.

Effects at the phylum and class levels

Figure 2 depicts changes at the phylum and class levels in the ileum, cecum, and feces microbiomes. Comparative analysis of phyla revealed that DSF decreased the median ilea levels of Actinobacteriota (−13%, male) and Firmicutes (−17%, female), which were compensated by elevations in the Firmicutes (+14%, male) and Bacteroidota (+16%, female). In the cecum and feces, the phyla changes did not have statistical significance (P > 0.05). At the class level, a greater number of taxa changes were discernible (Fig. 2 and Supplementary Fig. 2S). Enrichment of Bacilli was a common attribute in the cecum and feces of both sexes (P ≤ 0.06). Gammaproteobacteria were significantly depleted in the male ileum (log₂ FC −4.8, P < 0.0001) and cecum (−1.4, P < 0.0001) but elevated by 1.1-fold in the female ileum and feces. Other notable deviations at the class level included Clostridia (−1.8, P 0.007, female ileum), Bacteroida (+1.5, P 0.02, female ileum), Negativicutes (+7.1, P < 0.0001, male feces), Actinobacteria (+4.2, P 0.0002, male feces), and Fusobacteriia (+7.4, P 0.0002, male feces).

Figure 2.

Taxonomic distribution at the phylum level (left) and heatmap of scorers at the class level (right). The figures representing differential changes in mice treated with either vehicle (female = eight, male = eight) or DSF (female = eight, male = eight) indicate that DSF induces changes in Firmicutes (e.g. Bacilli, Clostridia) levels in the gut microbiome.

Effects at the family and genus levels

Figure 3 depicts the relative taxa changes at the family and genus levels. The female ileum showed notable depletion in members of the Clostridiaceae family (−31%) that was offset by enrichment of the Muribaculaceae (+16%) and Lachnospiraceae (+15%) populations. In male ilea, decreases in Enterobacteriaceae (−15%) and Streptococcaceae (−4%) were compensated by increases in the Lactobacillaceae (+7%) and Lachnospiraceae (+10%) populations. Comparably fewer alterations were detected at the family level in the ceca or fecal microbiomes. The most prominent was the elevation of Lactobacillaceae in both female and male mice (Supplementary Fig. 3S).

Figure 3.

Mean relative abundances at the family level (left) and heatmap of scorers at the genus level (right). The figures representing differential changes in mice treated with either vehicle (female = eight, male = eight) or DSF (female = eight, male = eight) indicate that DSF enriches Enterobacteriaceae and other Gram-negative enterics.

The heatmap in Fig. 3 further depicts the highest scorers at the genus level (Supplementary Fig. 4S). Most changes were associated with the male fecal microbiome (n = 29, P < 0.05). Among the 12 genera enriched by DSF were Aggregatibacter (+6.8, P 0.0006), Peptostreptococcus (+7.5, P 0.0003), Fusobacterium (+7.8, P 0.002), Prevotella (+7.9, P 0.0006), Parvimonas (+8.1, P 0.002), Hemophilus (+8.3, P < 0.0001), Neisseria (+9.1, P < 0.0001), and Porphyromonas (+9.5, P < 0.0001). The remaining scorers included depleted populations of Prevotellaceae UCG.001 (−1.1), Lachnospiraceae UCG.001 (−2.4, P 0.003), Candidatus Arthromitus (−4.4, P < 0.0001), and the Rikenellaceae RC9 gut group (−4.6, P < 0.0001). The female fecal levels of Prevotellaceae UCG.001 (−1.2, P 0.006) and the Rikenellaceae RC9 gut group (−1.9, P < 0.0001) were likewise altered, but not for Lachnospiraceae UCG.001 (+0.9, P 0.7) or Prevotella (−3.8, P 0.1). Collectively, DSF induced a moderate increase of Gram-positive population (+4%) in both female and male feces at the genus level.

In the cecum, Prevotellaceae UCG.001 (−2.3, P < 0.0001) and the Rikenellaceae RC9 gut group (−2.4, P < 0.0001) were also reduced by DSF in male mice. Other genera showing depletion in cecal samples were Streptococcus (−2.9, P < 0.0001, male), RF39 (−3.7, P 0.001, female), Enterococcus (−4.8, P 0.05, male), Escherichiahigella (−5.2, P 0.008, male), Acinetobacter (−6.8, P 0.0004, male), Psychrobacillus (−8.2, P < 0.0001, male), and Clostridium sensu stricto 1 (−8.9, P < 0.0001, male). Gram-type analysis showed that DSF increased the median Gram-positive microflora in the female cecum (+6%), but no deviation occurred in male mice (<0.1%) at the genus level.

In the ileum, the majority of genus-level changes took place in the female mice. Among those enriched were the Clostridia class members of Lachnoclostridium (+1.6, P 0.02), Marvinbryantia (+1.6, P 0.01), Eubacterium brachy group (+2.4, P 0.01), Eubacterium xylanophilum group (+1.8, P 0.007), Lachnospiraceae UCG.001 (+1.9, P 0.003), Lachnospiraceae FCS020 (+3.2, P 0.004), and A2 (+3.3, P < 0.0001). Notable depleted genera included Candidatus Arthromitus (−5.6, P < 0.0001) and Dubosiella (−6.2, P 0.001). The male ileum had comparably fewer scorers with only Escherichiahigella (−6.0, P 0.001) and Streptococcus (−3.9, P < 0.0001) of note. Collectively, DSF decreased the median female ileum Gram-positive microbiota by 17%, while the reverse effect was observed in male mice (+14%) at the genus level.

Effects at the species level

Species-level analysis revealed that DSF had the greatest influence on the cecal and fecal microbiomes (Supplementary Fig. 5S). About 70% of the 165 scorers (P < 0.05) were Gram-positive bacteria, many of which had an uncultured species designation. Most high scorers were present in the male feces and notably included Staphylococcus lentus (−6.4, P 0.03), Tannerella forsythia (+4.4, P 0.03), Filifactor alocis (+6.3, P 0.04), Fusobacterium periodonticum (+6.5, P 0.03), Porphyromonas gingivalis (+7.1, P 0.0005), Porphyromonas pasteri (+7.4, P 0.002), and Neisseria perflava (+8.6, P < 0.0001).

Figure 4 depicts Gram-positive species of the core murine gut microbiota depleted by DSF in both female and male mice. The obligate anaerobe Acutalibacter muris of Ruminococcaceae descent (Lagkouvardos et al. 2016) was depleted in male ileum, cecum, and feces. Clostridiales bacterium of the Lachnospiraceae NK4A136 group was likewise reduced in the male intestines, but the reverse effect was detected in female mice. The Gram-positive commensal Lactobacillus intestinalis was similarly enriched in female feces (+2.5, P 0.002) but depleted in the male cecum (−5.8, P < 0.0001). Another Lachnospiraceae relative, Eubacterium sp. of the Roseburia class (Mukherjee et al. 2020), was also decreased in both female cecum (−5.4, P 0.001) and feces (−4.3, P 0.01). Moreover, Streptococcus danieliae (Clavel et al. 2013) was comparably depleted by DSF in both the female ileum (−2.6, P 0.03) and male feces (−3.0, P 0.04).

Figure 4.

Differential analysis of core murine gut-associated species suggests that DSF treatment impacts the ileum and cecum microbiotas differently in female and male C57BL/6 mice. The bar graphs represent log₂ fold changes (FC) in mice treated with either vehicle (female = eight, male = eight) or DSF (female = eight, male = eight).

Discussion

The primary goal of this study was to determine the extent that Antabuse^® induces changes in the gut microbiota by differential analysis of the ileum, cecum, and feces. Prior research established that DSF has a narrow activity spectrum against Gram-positive species of the intestinal microflora akin to vancomycin (Citron et al. 2012). Similarly, Gram-negative bacteria, including Enterobacteriaceae, exhibit innate resistance to both vancomycin and DSF (Frazier et al. 2019). We therefore projected that DSF would enrich the Gram-negative microbiota and decrease alpha diversity in a manner similar to vancomycin (Zhang et al. 2022).

The susceptibility studies using isolates from the human gut corroborated that DSF inhibits the in vitro growth of the Gram-positive anaerobes, including Peptostreptococcus and Clostridium; however, commensal species of Bifidobacterium, Lachnospiraceae, and Ruminococcaceae exhibited low susceptibility to DSF, unlike vancomycin. Moreover, the Gram-negative Porphyromonas bivia was surprisingly susceptible to DSF as well as its metabolite DDTC. This implies that the chelating action of DDTC participates in the growth inhibition as observed for Porphyromonas gingivalis when treated with the Fe³⁺ chelator deferoxamine (Moon et al. 2011).

While the alpha diversity data indicate microbiota richness was not significantly impacted, it was apparent that DSF treatment altered the abundance of prominent commensal species (e.g. L. intestinalis). Lactobacilli are Gram-positive probiotic anaerobes that inhabit both the upper and lower intestines (Dempsey et al. 2022). The MICs obtained for L. paragasseri, L. jensenii, and L. johnsonii (>16 µg ml⁻¹) inferred that DSF would not deplete the Lactobacilli population. The in vivo data confirmed that the overall genus was slightly elevated in the female and male intestines as projected by the MIC data (Fig. 3). However, species-level analysis showed decreases in L. intestinalis levels throughout the male intestines (Fig. 4). The reduction appeared to be compensated by other Lactobacillaceae, which was elevated at the family level in both sexes. These data align with the prior observation that DSF enriches the fecal levels of Lactobacilli in male atherogenic mice (Traughber et al. 2024).

Similar to Lactobacilli, Clostridia are prominent residents of the intestines and believed to be influential on immunological and physiological gut health (Guo et al. 2020). Our study showed that Clostridia exhibit weak in vitro susceptibility to DSF that translated in vivo. Members of C. sensu stricto genera most affected as observed prior in the feces of nonalcoholic steatohepatitic (NASH) male mice treated with oral DSF (Lei et al. 2022). In this study, C. sensu stricto 1 (–9.0, P < 0.0001) was the most heavily depleted in the cecum for male mice. In female mice, DSF induced extensive decreases in the C. sensu stricto population, most prominently in the ileum. The apparent compensatory effect in the female ileum microbiome was enrichment of Muribaculaceae (+16%) and Lachnospiraceae (+15%). The mean 17% reduction in the Gram-positive population appeared to be countered by the elevation of Muribaculaceae (i.e. S24-7) consisting of Gram-negative, nonsporulating anaerobes (Lagkouvardos et al. 2019).

Alongside Clostridia, the Lachnospiraceae are considered beneficial microflora, but overgrowth has been correlated to human disease (Vacca et al. 2020). The MIC results indicated that Lachnospiraceae spp. are not appreciably susceptible to either DSF or DDTC. The in vivo data showed a 10%–15% enrichment of the Lachnospiraceae family in the ileum of both genders. Conversely, there was no significant deviation in fecal Lachnospiraceae, reflecting how the stool and intestinal tract data may not fully align when evaluating the global impact of a medication on the gut microbiome.

Other deviations to the core murine gut-associated taxa were further noted (Lagkouvardos et al. 2016, Beresford-Jones et al. 2022). The commensal anaerobe Acutalibacter muris of Clostridia descent was depleted by 2.5–5.6-fold in male mice (P ≤ 0.02). In female mice, DSF also decreased Eubacterium spp. of the Clostridia taxa by 4.3–5.4-fold (P ≤ 0.04). Most Streptococcus spp. were likewise depleted as predicted by prior reported MICs (Frazier et al. 2019). Overall, the inhibitory effects of DSF on the Gram-positive flora were most evident in the female ileum and compensated by enrichment of Gram-negative Bacteroidota members (i.e. Muribaculaceae). Significant elevations of certain Gram-negative genera were also observed in male feces. High scorers with a log₂ FC > 6.5 included Aggregatibacter, Fusobacterium, Porphyromonas, and Neisseria spp. The collective data therefore indicate that DSF reduces the abundance of Gram-positive enterics and enriches the Gram-negative population. In the final analysis, it was concluded that DSF induces substantive changes in the gut microbiome that can impact intestinal health when prescribed for a long term as a maintenance drug.

The regulatory approval of Antabuse^® in 1951 has improved the quality of life for many persons with alcohol dependence. In the decades since 1951, research has revealed DSF to be a promiscuous drug with a complex pharmacology that includes antimicrobial activity against bacteria and yeast (Custodio et al. 2022, Lanz et al. 2023). The daily 125–500 mg regimens of DSF expose the gut microbiota to large quantities of the drug for a period of months to years. This can result in gastrointestinal disturbances precipitated by depletion of beneficial commensals that are susceptible to DSF and its metabolites. When this occurs, probiotics may aid in restoring the physiological and immunological health of the gut microbiome while impairing colonization by pathogenic species. Moreover, naltrexone (Trexan^®) and acamprosate (Campral^®) are available as alternative medications approved by regulatory agencies for alcohol use disorder should an intestinal disorder arise with DSF (Kranzler 2023).

Author contributions

Sarah E. Evans (Data curation, Investigation, Writing—review & editing), Meagan E. Valentine (Data curation, Investigation, Writing—review & editing), Fallon Gallimore (Data curation), Yogesh Meka (Data curation), Samuel I. Koehler (Formal analysis, Writing—review & editing), Hongwei D. Yu (Funding acquisition, Methodology, Supervision, Writing—review & editing), Monica A. Valentovic (Funding acquisition, Methodology, Writing—review & editing), and Timothy E. Long (Conceptualization, Formal analysis, Funding acquisition, Methodology, Project administration, Supervision, Visualization, Writing—original draft)

Conflict of interest

None declared.

Funding

This research was supported by the National Institute of Allergy and Infectious Diseases, and National Institutes of Health grants AI151970 and P20GM103434 to the West Virginia IDeA Network of Biomedical Research Excellence.

Data availability

The data underlying this article are available in the article and in its online supplementary material.