Antibiotics Can Reshape Host Immunity and Circadian Rhythms Without Any Microbes

For nearly a century, antibiotics have been understood as weapons aimed squarely at bacteria. Their effects on the body were assumed to flow through one channel: kill or perturb the gut microbiota, and the host responds to whatever the microbes no longer do. A new study published in Nature Microbiology by a team at Bar-Ilan University and the Hebrew University of Jerusalem now shows that this microbiota-only model is incomplete. Using a combination of germ-free mice, metagenomic sequencing, transcriptomics and a purpose-built computational tool called CompoRes, the researchers demonstrate that vancomycin, one of the most widely used antibiotics in clinical practice, directly rewires host physiology in ways that persist even when no microbes are present at all.

The central challenge the team confronted is one that has long frustrated microbiome researchers. When an antibiotic treatment changes both the composition of the gut microbiota and the expression of host genes, it is extraordinarily difficult to determine which host changes are caused by the microbial shifts and which are caused by the drug acting directly on host cells. Germ-free animals can reveal whether a perturbation acts directly on the host, but until now there was no rigorous statistical tool that could predict, from observational data in conventional animals, whether a given host response was microbiota dependent or microbiota independent. The researchers set out to close that gap.

Their experimental design was ambitious. They treated specific pathogen-free mice with four commonly used antibiotics—vancomycin, ampicillin, metronidazole and neomycin—either individually or in combination, and delivered them through three different routes: oral gavage, intraperitoneal injection and intravenous injection. Mice were treated twice daily for three and a half days, mimicking the shorter antibiotic regimens now favoured in clinical practice. Stool samples were collected before and after treatment to profile the microbiota using 16S rRNA amplicon sequencing, while colonic tissue was harvested for RNA sequencing to capture the host transcriptional response.

One of the most surprising findings concerned vancomycin itself. This is a large, hydrophilic molecule that should cross the gut mucosa poorly, which is precisely why it is given orally to treat intestinal infections like Clostridioides difficile. Yet when the researchers administered vancomycin systemically by intraperitoneal injection, mass spectrometry confirmed that detectable amounts of the drug reached the small intestinal and colonic lumen. The consequences for the microbiota were distinct depending on the route: oral administration drove a dramatic increase in Enterobacteriaceae, while systemic treatment increased Tannerellaceae. Both are Gram-negative families, but the fact that systemic vancomycin reshapes the gut community at all overturns a long-standing assumption about the drug’s pharmacokinetics.

When the team turned to the host transcriptome, the picture became even more interesting. Intraperitoneal vancomycin affected the largest number of colonic transcripts of any antibiotic studied, and gene set enrichment analysis revealed that the affected pathways included antigen presentation via major histocompatibility complex class II molecules—a core immune function of the gut epithelium. A random forest classifier trained on the transcriptional data could distinguish each antibiotic and each administration route with high accuracy, confirming that different antibiotics produce unique host transcriptional signatures. Remarkably, when the four antibiotics were combined, many of the individual effects on core clock genes were suppressed, and even clinically relevant pairs such as vancomycin plus metronidazole produced non-additive effects on the host that could not be predicted from the single-drug responses.

To disentangle microbiota-dependent from microbiota-independent effects, the researchers developed CompoRes, a computational tool that integrates microbial compositional data with host gene expression profiles. The algorithm first infers correlations among microbial taxa using SPARCC, a method designed for compositional data, and hierarchically clusters them into operational compositional units. It then computes log-ratios of all possible pairs of these units and correlates them with gene expression. Because the number of comparisons is vast relative to sample size, the tool generates a null distribution by shuffling the data at least fifty times, yielding a rigorous P value for each gene. The approach was validated on synthetic data and on experimental ground truths: germ-free mice treated with intraperitoneal vancomycin, where the drug can only act directly on the host, and germ-free mice receiving a faecal microbiota transplant from orally treated mice, where the effect can only flow through the microbiota.

Applying CompoRes to the clock genes Ciart, Nfil3, Per3 and Dbp revealed a striking route-dependent dichotomy. In mice treated orally with vancomycin, the expression of these core circadian regulators correlated strongly with changes in microbiota composition, consistent with previous work showing that the gut microbiota oscillates in a circadian pattern and programs host transcriptome oscillations. But in mice receiving the same drug systemically, the same genes showed no correlation with the microbiota whatsoever. The tool had predicted, purely from data in conventional animals, that systemic vancomycin disrupts circadian gene expression in the gut through a direct, non-microbial pathway. Subsequent experiments in germ-free mice confirmed this prediction: intraperitoneal vancomycin altered core clock gene expression across a 24-hour cycle in animals that had no microbes at all.

The physiological consequences of this microbiota-independent disruption proved to be behavioural. In germ-free mice, vancomycin reversed the normal diurnal expression pattern of metabolic genes such as Ppara, Cd36, Lpl and Fabp4, which typically peak during the light phase when nocturnal mice are fasting. The team found that vancomycin reduced food intake in both germ-free and conventional mice, with feeding normalising after treatment ceased. Transcriptional analysis showed that the drug uniquely altered enteric nervous system transcripts, and immunofluorescence imaging revealed that fluorescein-labelled vancomycin was internalised into myenteric plexus neurons following systemic injection but not oral administration. The drug induced expression of the neuropeptide neuromedin U and its receptor NMUR2, and mice lacking neuromedin U were resistant to vancomycin-induced hypophagia, pinpointing the molecular mechanism by which the antibiotic suppresses appetite.

The downstream immune consequences were equally profound. Expression of the MHC class II genes H2-Aa and H2-Ab1 in the colonic epithelium normally peaks at a specific circadian time point, and the researchers discovered that this oscillation persists in germ-free mice—unlike in the small intestine, where microbial presence is required. They further showed that glucose directly induces H2-Ab1 expression in fasting germ-free mice and in intestinal epithelial cells in vitro, indicating that colonic MHC class II expression follows a diurnal, glucose-responsive pattern dictated by feeding. Vancomycin-induced hypophagia suppressed this peak entirely, meaning the antibiotic disrupts a key antigen-presentation rhythm in the gut through a direct behavioural pathway rather than through any change to the microbiota.

Perhaps most remarkably, the molecular response to vancomycin proved to be evolutionarily conserved. When the researchers reared Drosophila melanogaster on vancomycin-laced food and sequenced adult fly brain RNA, they found that the fly homologues of the mammalian Clock gene and its downstream target timeless were altered, as was klumpfuss, a gene controlling feeding behaviour in flies. The fly orthologue of the NMU receptor was induced in response to the drug, mirroring what the team had observed in mice. This preservation of the vancomycin response across evolutionarily distant organisms suggests that the antibiotic taps into an ancient cellular stress pathway, one that links metabolism, circadian biology and immunity through mechanisms that operate independently of any microbe. The work challenges the microbiota-only model of antibiotic action and provides a framework for identifying direct effects of environmental perturbations on host physiology, with implications for understanding how antibiotics shape immunity, metabolism and circadian health in clinical practice and beyond.

Subject of Research: Microbiology

Article Title: Disentangling microbiota-dependent and -independent impacts of antibiotics on host physiology

Article References: Zelik, L., Levin, Y., Sawaed, J., Telpaz, S., Zigdon, M., Nuriel-Ohayon, M., Rubin, E., Gordon, A., Mogilevski, Z., Yehuda, A., Awad, A., Shaalan, H., Ben-Simon, S., Modilevsky, S., Naama, M., Dalvi, J., Moadi, L., Kurtz, M., Perez, S., … Bel, S. (2026). Disentangling microbiota-dependent and -independent impacts of antibiotics on host physiology. Nature Microbiology. https://doi.org/10.1038/s41564-026-02453-w

Image Credits: AI Generated

DOI: 10.1038/s41564-026-02453-w

Keywords: antibiotics, gut microbiota, circadian rhythms, vancomycin, MHC class II, neuromedin U, germ-free mice, CompoRes, host physiology, microbiome, Disentangling, microbiota-dependent