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Published in final edited form as: Science. 2026 Sep 3;393(6815):1045–1052. doi: 10.1126/science.ady2886

Early-life colonization with Clostridioides difficile remodels the developing gut

Alexa Semon 1,2,3,†, Haider S Manzer 1,2,4,†, Orlaith Keenan 1,2,5, Tiffany H Zhou 1,2,5, Connor Tiffany 1,2,4, Qianxuan She 1,2,4,5,6,7, Montana Knight 8, Ashley S Weiss 1,2,5, Kassy Donohoe 1,2, Brendan T Morrow 9,10, Rochelle C Glover 1,2,4, Maria E Kovalik 3, Ceylan Tanes 4,6, Kyle Bittinger 4,6,7, Babette S Zemel 6,7, Gary D Wu 11, Michael A Silverman 4,7,12, Jeffrey S Gerber 10,12, Rubén Cano Rodríguez 13, D Borden Lacy 13,14,15, Tatiana A Karakasheva 6,16, Mohamad-Gabriel Alameh 1, Drew Weissman 17, Stacey L Schultz-Cherry 9, Jason W Rosch 9, Paul J Planet 4,7,12,18, Ahmed M Moustafa 4,6,7, Emma E Furth 19, Michael C Abt 3,5, Judith R Kelsen 6,7, Maire A Conrad 4,6,7, Kathryn E Hamilton 6,16, Joseph P Zackular 1,2,3,4,*
PMCID: PMC13626140  NIHMSID: NIHMS2206797  PMID: 42691172

Abstract

Clostridioides difficile causes severe disease in adults but commonly colonizes infants asymptomatically. The consequences of early-life colonization on host development remain unknown. In a neonatal mouse model, C. difficile colonization drove pro-inflammatory and tissue repair responses in the intestinal epithelium, enriching injury-associated intestinal stem cell populations and skewing differentiation toward secretory lineages. Despite transient colonization, exposure to C. difficile early in life led to persistent changes into adulthood. Epithelial responses were toxin-dependent, as colonization with non-toxigenic strains or maternal vaccination with a C. difficile-targeted mRNA-LNP vaccine protected neonates. Human infant intestinal epithelial cells were sensitive to C. difficile toxins, and biopsies from colonized infants exhibited altered intestinal stem cell behavior. This study redefines C. difficile as an underappreciated early-life pathogen with lasting effects on host development.

One-Sentence Summary:

Asymptomatic colonization with C. difficile early in life reshapes the developing gut.

The intestinal microbiota plays a critical role in influencing host immunity and maintaining gut homeostasis, particularly during early life, when microbial colonization coincides with the mucosal immune system development (1). Timing of this relationship is crucial to establishing immune tolerance to commensal microbes while maintaining the capacity to combat pathogens. Disturbance to this ecosystem early in life can increase susceptibility to disorders later in life (1–5).

Among early-life taxa, Clostridioides difficile is notable due to its role as both a common colonizer of the neonatal intestine and a prominent adult enteric pathogen (6–11). Up to 70% of infants are reported to be colonized with C. difficile; however, colonization is clinically asymptomatic, suggesting distinct early-life host-microbe interactions (8, 12–16). This dichotomy between infant and adult outcomes has led to hypotheses that neonatal tolerance is mediated by immature toxin receptors (17) or maternal antibodies (18). However, both mechanisms have been challenged by studies showing that toxin binding kinetics (19) and feeding method do not fully explain asymptomatic colonization (12). The assumption that asymptomatic colonization is benign has obscured the consequences of colonization with C. difficile during this critical early-life developmental window. In this study, we provide evidence that toxigenic C. difficile reshapes neonatal development and has lasting consequences on the host.

C. difficile is highly prevalent in infants

Previous studies have reported that asymptomatic colonization with C. difficile occurs in 40–70% of infants during the first two years of life (8, 12–16). Indeed, in a cohort of infants born in Philadelphia, Pennsylvania, we found that 28.2% were colonized by C. difficile within the first month of life, and nearly 75% by two years (Fig. 1A). Within these infants, C. difficile represents a minor proportion of the total microbiome, comprising an average of 0.43% at 24 months (Fig. 1B). Genomic analysis of C. difficile strains isolated from infants (<1 year) revealed expression of toxin genes and phylogenetically clustering with strains isolated from children (2–18) and adults (18+) with active infection (fig. S1A) (12, 20–23).

Figure 1. Asymptomatic colonization of C. difficile is associated with transcriptional changes in neonatal IECs.

Figure 1.

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C. difficile prevalence (A) and relative abundance (B) in stool samples from healthy infants over first 24 months of life. (A – B) n = 106 (1 month), 139 (4 months), 133 (12 months), and 87 (24 months). B is represented as percentage of total reads. (C – K) Neonatal mice were orally gavaged on post-natal day 8 (P8) with 100,000 spores of C. difficile VPI 10463. Adult mice were treated with cefoperazone in their drinking water for 5 days, followed by two days of recovery on normal water, and infected with 1,000 spores of C. difficile VPI 10463. (C) Survival curve. n = 6 per group. Representative images (D) and pathology scoring (E) of ceca stained with hematoxylin and eosin in C. difficile (+Cd)-infected adults (AD), +Cd P14 neonates, and untreated age-matched controls. Scale bar, 250 μm. n = 5 mice per group. (F) Fecal lipocalin-2 levels in untreated (NT, white) and C. difficile-colonized (Cd, green) P14 neonates. n = 4 – 5 mice per group. (G) Intestinal epithelial cell (IEC) viability in age-matched NT and Cd P14 neonates. n = 27 per group. (H) Volcano plot of RNA-seq on IECs from C. difficile-colonized neonates and untreated neonatal controls. Red dots represent genes passing multiple test corrected significance threshold (FDR p-value <= 0.05) and absolute logFC > 1. (I) Gene set enrichment analysis (GSEA) of the neonatal RNA-seq results. Bars show normalized enrichment scores (NES) of the significant (FWER p-value < 0.05) hallmark gene sets from GSEA. Color indicates enriched in Cd (green) or NT neonate (black). Representative images (J) and quantification (K) of Ki67 staining of cecal tissues from untreated (NT) or C. difficile (Cd) colonized P14 neonates. Scale bar, 200 μm. n = 6 – 8 mice per group.

Data are represented as mean ± SEM and are representative of 2–3 independent experiments. Each dot represents an individual human (B) or individual mouse (F, G, K). Statistics by Log-rank (Mantel-Cox) test (C), one-way ANOVA with Tukey’s multiple comparisons test (E), unpaired t test (F, G, K). ns (not significant) p >0.05, *p <0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Neonatal mice are protected from C. difficile infection-associated mortality

To systematically define the impact of early-life exposure to C. difficile on the host, we established a neonatal mouse model that recapitulates asymptomatic colonization observed in human infants (Fig. 1C and fig. S2A – C). Neonatal mice were susceptible to C. difficile colonization without antibiotic perturbation of the mother or neonates, resulting in comparable burdens observed in established adult murine models of infection (fig. S2B, C) (24). Following oral inoculation with 100-times the adult lethal dose of C. difficile (VPI 10463), neonatal mice remained fully protected against morbidity and mortality (Fig. 1C). C. difficile burdens and toxin titers remained stable in neonates during early life, falling below the limit of detection after weaning (fig. S2B, C). Furthermore, neonatal colonization with C. difficile did not significantly impact the succession or composition of the early-life microbiota (fig. S2D – H, and supplemental table 1).

C. difficile produces two exotoxins, TcdA and TcdB, which drive disease pathology in adults. While neonatal mice exhibited robust resistance to symptoms of disease during C. difficile colonization, systemic recombinant C. difficile toxin B (rTcdB) challenge resulted in 100% mortality within 24 hours (fig. S2I), suggesting that protection is conferred by local rather than systemic mechanisms.

Recent work has implicated IL-17-producing γδ T cells as key mediators of neonatal protection from C. difficile-associated mortality (25). Notably, the published model used broad-spectrum antibiotic perturbation to sensitize neonates to C. difficile colonization, raising the question of whether γδ T cells play a similar protective role in an antibiotic-free setting. To test this, we colonized recombination-activating gene 1 (RAG1) knockout neonates, which lack B cells and T cells including γδ T cells, and found that they remained protected from C. difficile-associated mortality, suggesting that additional mechanisms contribute to neonatal protection in our model (fig. S2J).

Neutrophil counts in blood and subsequent infiltration into the large intestine are major correlates of disease severity in infected adults (26). Thus, we reasoned that neutrophil recruitment may be stunted in neonates following C. difficile colonization. Neonates did not show neutrophilic expansion in the blood or infiltration into the large intestine in response to C. difficile, which was in stark contrast with adults (Fig. 1D – E and fig. S2K). Pharmacological reduction of neutrophil recruitment to the intestine during C. difficile infection markedly reduces disease severity in adult mice (27). Taken together, these data suggest that the absence of neutrophil expansion and recruitment contributes to protection from symptomatic disease in neonates, but the underlying mechanism is likely multifaceted.

C. difficile elicits an inflammatory response in neonatal intestinal epithelial cells

Despite the absence of gross pathology, we found that neonatal mice colonized with C. difficile had elevated lipocalin-2 levels in their stool, indicating an activated inflammatory response (Fig. 1F). Additionally, intestinal epithelial cells (IECs) isolated from infected neonates exhibited decreased viability and increased expression of cleaved Caspase-3, suggesting an uncoupling of inflammation and pathological damage (Fig. 1G and fig. S2L, M). Thus, to gain a mechanistic understanding of the neonatal IEC response to C. difficile, we performed bulk RNA sequencing (RNA-seq) on IECs isolated from C. difficile colonized neonates at P14 (day 6 post-inoculation) and age-matched controls (Fig. 1H, I and fig. S3A). We identified 72 genes to be significantly differentially expressed upon C. difficile colonization, of which 67 were upregulated and 5 were downregulated (Fig. 1H). Enriched transcripts were involved in inflammatory immune responses such as, Ly6g, Ly6d, Ly6a, Cxcl5, and Il18 and anti-microbial responses including, Reg3g, Slpi, Ltf, and Lcn2 (Fig. 1H and fig. S3B). Cytoskeletal related genes including Arhgap20 and Cyria were downregulated in the presence of C. difficile (Fig. 1H). A subset of neonatal differentially expressed genes (DEGs) showed no clear pattern in expression dynamics over time (fig. S3B).

Gene-set enrichment analysis (GSEA) revealed an enrichment in interferon-gamma (IFNγ) and IFNɑ gene signatures, along with other inflammatory response signatures including tumor necrosis factor-alpha (TNFɑ) signaling via NF-kB and IL6/JAK/STAT3 activation in C. difficile colonized neonates (Fig. 1I). Elevated phosphorylated STAT3 levels and increased expression of Il22 in IECs isolated from C. difficile colonized neonates are consistent with an active epithelial regeneration response (fig. S3C–E) (28, 29). Towards that end, GSEA also revealed gene signatures related to damage responses, cell cycle, and proliferation, including G2M checkpoints, E2 promoter binding factor (E2F) targets, and MTORC1 signaling (fig. S3B). Ki67 staining confirmed increased proliferation at the base of epithelial crypts in colonized neonates (Fig.1 J, K).

We next performed a comparative analysis between the neonatal epithelium-specific transcriptional response to C. difficile colonization and a previously published whole-colon adult gene signature observed during C. difficile infection (fig. S3F – K) (30). We found that neonatal DEGs highly intersected with DEGs observed in acute adult C. difficile infection timepoints (AD3–5), with correlation decreasing after recovery (AD8–10) (fig. S3F – I). Furthermore, GSEA revealed that neonates share similar pathway responses as infected adult mice (fig. S3J, K).

Early-life C. difficile colonization alters neonatal intestinal stem cell behavior

Given the enrichment of proliferation and regeneration signatures in C. difficile colonized neonatal IECs, we next sought to assess intestinal stem cell (ISC) function directly. ISCs were isolated from C. difficile-colonized and untreated neonatal mice at P14 and assessed for their ability to develop colonic organoids as a readout of ISC functionality and regenerative potential (Fig. 2A, B). We observed that ISCs from neonatal mice colonized with C. difficile exhibited an increased organoid forming efficiency in cultures compared to their age-matched controls (Fig. 2A, B). Organoids from C. difficile-colonized neonates had altered growth behaviors, including an increase in eccentricity and budding (Fig. 2A, B), indicative of enhanced regenerative potential and differentiation (31).

Figure 2. Early life C. difficile leads to enrichment of regenerative ISC populations and secretory cell markers.

Figure 2.

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Neonatal mice were colonized with C. difficile (Cd, green) on post-natal day 8 (P8). Age-matched neonates served as untreated controls (NT, white). IECs and tissues were harvested on P14. (A) Representative brightfield images with 4x magnification of organoids 7 days post-plating. Scale bar, 2 mm. (B) Quantification of organoid forming efficiency (OFE) shown as percent of total number of cells plated, organoid size represented as arbitrary units (AU), likeness to a perfect circle (eccentricity), and the percent of organoids displaying budding morphology. n = 13 per group. (C) Quantification of stem cell markers on IECs by RT-qPCR. n = 4 – 9 per group. (D) Quantification of stem cell frequencies measured by flow cytometry. n = 5 – 7 per group. (E) Quantification of differentiated cell markers on IECs by RT-qPCR. n = 8 – 9 per group. Representative images from immunofluorescence microscopy (F) and quantification (G) of cells expressing secretory cell markers. DAPI (gray), MUC2 (green) DCLK1 (red), and CHGA (magenta). Scale bar, 200 μm. n = 10 – 12 per group.

Data are represented as mean ± SEM or violin plot and are representative of 3 independent experiments. Each dot represents an individual mouse (B – E, G). Statistics by unpaired t test (B – E, G). ns (not significant) p >0.05, *p <0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Leucine-rich repeat-containing G protein-coupled receptor 5-positive (LGR5+) and stem cell antigen 1-positive (SCA-1+; Ly6a expressing) stem cells represent two ISC subsets, each with unique roles in epithelial maintenance and regeneration. LGR5+ ISCs drive homeostatic renewal of the intestinal epithelium, whereas SCA-1+ ISCs mediate injury-induced regeneration (31–33). Ly6a was among the most upregulated genes in IECs from colonized neonates, while Lgr5 expression was unchanged (Fig. 1H, Fig. 2C). Flow cytometry confirmed that SCA-1+ ISCs were enriched in neonates colonized with C. difficile (Fig. 2D).

Furthermore, we found that early-life C. difficile led to increased expression of secretory cell lineage markers, including Dclk1 (tuft cells), Muc2 (goblet cells), and Chga (enteroendocrine cells), without corresponding changes in the absorptive colonocyte marker Apob (Fig. 2E). Fluorescent microscopy confirmed that Mucin 2-positive (MUC2+) goblet cells were enriched in neonates colonized with C. difficile (Fig. 2F, G). Critically, these changes persisted into adulthood. 8-week-old adult mice that had been previously colonized with C. difficile as neonates (C. difficile pre-col) showed an expansion of MUC2-expressing goblet cells and a concurrent loss in doublecortin-like kinase 1 (DCLK1) -expressing tuft cells in vivo compared to age-matched control mice, despite clearance of C. difficile after weaning (Fig. 3A, B, and fig. S2B to C). Moreover, we found that after 10 ex vivo passages, ISCs from C. difficile-colonized neonates retained their augmented growth potential and organoid morphology observed after initial plating (Fig. 3C, D), and we observed trends towards increased expression of Muc2 and Chga compared with controls, suggesting a cell-intrinsic mechanism of altered differentiation patterns (Fig. 3E). Furthermore, expression of Ly6a remained high in serially passaged organoids, and Lgr5 expression decreased (Fig. 3F). Finally, in vivo lineage tracing demonstrated that canonical LGR5+ ISC differentiation drives goblet cell expansion in response to C. difficile (fig. S4A) (34, 35). Taken together, these data suggest that C. difficile elicits two parallel responses in the neonatal epithelium, with the emergence of injury-repair ISCs driving epithelial regeneration and concurrent reprogramming of LGR5+ ISC differentiation.

Figure 3. Epithelial behaviors following early life C. difficile are stable and persist through adulthood.

Figure 3.

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(A – B) Neonatal mice were colonized with C. difficile on P8 and allowed to age to 8 weeks of life (Cd pre-col, green). Age-matched mice served as untreated controls (NT, white). Representative images from immunofluorescence microscopy (A) and quantification (B) of cells expressing secretory cell markers. DAPI (gray), MUC2 (green) DCLK1 (red), and CHGA (magenta). Scale bar, 250 μm. n = 6 – 7 mice per group. (C – E) Neonatal mice were colonized with C. difficile (Cd, green) on post-natal day 8 (P8). Age-matched neonates served as untreated controls (NT, white). On P14, colonic crypts were harvested, digested into single cells, and plated in Matrigel. After 7 days in culture, organoids were dissociated into single cells and passaged for a series of 10 passages. (C) Representative brightfield images with 4x magnification of organoids after 10 passages. Scale bar, 2 mm. (D) Quantification of organoid forming efficiency (OFE) shown as percent of total number of cells plated, organoid size represented as arbitrary units (AU), likeness to a perfect circle (eccentricity), and the percent of organoids displaying budding morphology at passage 10. n = 4 – 5 mice per group. RT-qPCR for differentiated cell (E) and stem cell (F) markers on organoids derived from Cd and NT ISCs after 10 passages. (G) Log10 murine astrovirus (MuAstV) genome copies on day 12 post-viral infection in 8-week-old Cd pre-col and age-matched controls. n = 12 – 13 mice per group.

Data are represented as mean ± SEM or violin plot and are representative of 2 independent experiments. Each dot represents an individual mouse. Statistics by unpaired t test. ns (not significant) p >0.05, *p <0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

We next sought to examine whether long-term effects of C. difficile on the host epithelium would impact susceptibility to adult infection. Goblet cells can serve as replication niches for enteric viruses, including murine astrovirus (muAstV). C. difficile pre-col adult mice had increased viral burden in their stool 12 days post-viral infection compared to controls (Fig. 3G). We also found that early-life pre-colonization did not confer protection against, nor increase susceptibility to adult C. difficile infection-associated weight loss or epithelial damage (fig. S4B, C). Furthermore, while pre-colonization resulted in similar hyperproliferation and differentiation phenotypes observed at P14, adult C. difficile infection resulted in decreased organoid formation regardless of pre-colonization (fig. S4 D, E). These findings are consistent with previous reports that adult C. difficile infection results in damage to ISCs and impaired epithelial recovery (36).

Neonatal C. difficile colonization reduces innate lymphoid cell populations

Given the intimate crosstalk between epithelial and immune compartments, we next examined whether these epithelial changes were accompanied by alterations in mucosal immune populations.

Immunophenotyping of colonic lamina propria immune cells in C. difficile pre-col mice and age-matched controls at 4 and 8 weeks of life revealed significant reductions in the frequencies of GATA3+ type 2 innate lymphoid cells (ILC2) and retinoic acid-related orphan receptor gamma t (RORγt)+ ILC3s in C. difficile pre-col mice (fig. S5A – D). These reductions occurred independently of changes in T helper (Th) cell subsets (fig. S5E) or myeloid cell populations (fig. S5F, G). Furthermore, long-term immune changes in C. difficile pre-col mice were not driven by persistent microbiota dysbiosis (fig. S6 and supplemental tables 2, 3). Although, it is possible that transient shifts in composition or functional differences may contribute to these phenotypes.

Early-life intestinal damage is driven by C. difficile toxins

To gain a mechanistic understanding of how C. difficile impacts neonatal IEC behavior, we tested whether epithelial responses were dependent on C. difficile toxin or whether colonization was sufficient. First, we treated neonatal IECs with recombinant TcdB in vitro, and found toxin treatment was sufficient to significantly decrease IEC viability (fig. S7A). Next, we colonized mice with either toxigenic or non-toxigenic strains of C. difficile isolated from healthy human infants. Both clinical strains colonized the neonatal GI tract to similar burdens as VPI 10463 (fig. S7B), and toxin was detected at comparable titers in the feces of neonates colonized with the toxigenic clinical strain (fig. S7C). Neonatal mice were protected from infection with clinical C. difficile isolates (fig. S7D). Compared to untreated controls, IECs from neonates colonized with the toxigenic strain of C. difficile had reduced cell viability and increased expression of top DEGs from the RNA-seq, including Lcn2, Reg3g, and Slpi, which was not observed in IECs from neonates colonized with non-toxigenic C. difficile (fig. S7E, F). Organoids formed from colonic ISCs isolated from neonatal mice colonized with the toxigenic clinical isolate also displayed increases in organoid-forming efficiency, eccentricity, and budding (fig. S7 G, H). Furthermore, these phenotypes were consistent with ISCs isolated from neonates colonized by wildtype C. difficile strain R20291, but not an isogenic ΔtcdAΔtcdB mutant (fig. S7 I, J). Long-lived organoid phenotypes, assessed across 10 serial ex vivo passages, were only observed in toxin-producing C. difficile strains (fig. S8A, B). Maintenance of goblet cell and enteroendocrine cell hyperplasia (fig. S8 C, D) as well as decreases in ILC populations (fig. S8E) into adulthood were dependent on neonatal colonization specifically by toxigenic C. difficile. These data collectively demonstrate that neonatal IEC responses to C. difficile colonization are driven by the effects of toxin on ISCs rather than pathogen colonization alone.

Maternal vaccination protects against toxin-dependent effects of early-life C. difficile

C. difficile-colonized mothers are capable of transferring anti-C. difficile antibodies through cord blood and breast milk (37). To investigate whether passive immunization could protect the neonatal gut against C. difficile-mediated damage, we adapted a multivalent mRNA-LNP C. difficile vaccine for use in a maternal vaccination strategy (38). Maternal vaccination resulted in detectable anti-TcdA and anti-TcdB IgG and IgA antibodies in the gastric contents and stool of the neonates (Fig. 4A, B). Although antibody titers did not reduce intestinal C. difficile burden, colonized neonates born to vaccinated dams had reduced fecal toxin titers, increased IEC viability, and attenuated hyperproliferation as measured by Ki67 staining (Fig. 4C – G). ISCs harvested from C. difficile-colonized neonates born to vaccinated dams grew into organoids that were phenotypically similar to those from uncolonized control neonates, demonstrating a rescue of C. difficile-mediated hyperproliferation and aberrant differentiation (Fig. 4H, I). These antibody-mediated protective effects were durable, as subsequent litters from vaccinated dams remained protected (fig. S9).

Figure 4. Maternal vaccination protects against early life C. difficile-mediated damage.

Figure 4.

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Female mice were immunized with C. difficile mRNA-LNP vaccine. Unvaccinated dams served as negative controls. Neonatal mice born to vaccinated dams (Vax) or unvaccinated controls (Naive) were colonized with C. difficile on P8 and analyzed at P14 (Cd, green). Age-matched neonatal mice served as untreated controls (NT, white). Anti-TcdA/B IgG (black) and IgA (white) titers from the stomach contents (A) and (B) stool of neonates born to vaccinated dams were measured by ELISA. No antibodies were detected in neonates born to unvaccinated dams. n = 23 – 26 mice per group. Values of zero indicate antibody was not detected; dotted lines indicate assay limit of detection (LoD). C. difficile colony forming units (CFU) (C) and toxin titers (D) in neonatal stool. n = 13 – 15 mice per group. (E) Colonic epithelial cell viability. n = 11 – 15 mice per group. Representative images (F) and quantification (G) of Ki67 staining of cecal tissues neonates. n = 7 – 8 mice per group. (H) Representative brightfield images with 4x magnification of organoids 7 days post-plating. Scale bar, 2 mm. (I) Quantification of organoid forming efficiency (OFE) shown as percent of total number of cells plated, organoid size represented as arbitrary units (AU), likeness to a perfect circle (eccentricity), and the percent of organoids displaying budding morphology. n = 11 – 15 mice per group.

Data represented as mean ± SEM and representative of 2 independent experiments. Each dot represents an individual mouse. Statistics by unpaired t test (C, D) or two-way ANOVA with Tukey’s multiple comparisons test (E, G, I). ns (not significant) p >0.05, *p <0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

C. difficile colonization impacts human infant IECs

To examine the relevance of these findings in humans, we first noted that stool from C. difficile-colonized human infants had elevated lipocalin levels, consistent with our murine model (Fig. 1F and fig. S10A). To explore the effects of C. difficile on the human intestinal epithelium, we acquired colonic biopsies from five human infant patients, along with paired stool samples to determine C. difficile colonization status (supplemental table 4). RNA isolated from the biopsy of one C. difficile colonized infant displayed elevated Ly6a, Muc2, and Chga, with no changes in Lgr5, Dclk1, or Apob relative to a non-colonized infant (Fig. 5A, B). C. difficile toxin treatment of human infant IECs caused dose-dependent cell death and decreased barrier function (Fig 5C, and fig. S10B – D). Organoids derived from two C. difficile-colonized human infants displayed increased proliferation and differentiation across multiple passages, similar to results observed in neonatal mice (Fig. 5D, E). RNA isolated from these organoids also displayed a shift towards increased Ly6a, Muc2, and Chga expression across multiple passages (fig. S10 E, F). These data suggest a direct effect of C. difficile toxin on human infant ISCs and challenge the assumption that infant IECs are inherently resistant to C. difficile toxins, although the limited sample size warrants further validation in future studies.

Figure 5. Human infants are susceptible to early life C. difficile-mediated damage.

Figure 5.

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RNA was harvested from primary human infant intestinal epithelial cells isolated from colonic biopsies from C. difficile-negative (Infant A) and C. difficile-positive (Infant C) patients for RT-qPCR assessment of stem cell (A) and differentiated cell (B) markers. n = 1 infant with 3 technical replicates per group. (C) 2-D primary infant IEC cultures were treated with rTcdB (shades of red) or vehicle (Mock). Data is represented as LDH release normalized to Mock (0% death) and Triton X-100 (100% death) treatment groups. n = 3 passages per group. (D) Primary infant ISCs were used to seed organoid cultures. Representative brightfield images with 4x magnification of organoids 12 days post-plating. Scale bar, 2 mm. (E) Quantification of organoid forming efficiency (OFE) shown as percent of total number of cells plated, organoid size represented as arbitrary units (AU), likeness to a perfect circle (eccentricity), and the percent of organoids displaying budding morphology. n = 3 passages per group. (F) Map of countries sampled. Color indicates categorization of industrialized (red), transitional (blue), non-industrialized (green), or not sampled (white) communities. The number of samples per category is shown in the pie chart. (G) Prevalence of C. difficile in infants across lifestyles over the first 2 years of life. An abundance threshold was set at 0.1%. (H) C. difficile relative abundance across lifestyles during the first 2 years of life. Interpolation was performed with a non-parametric regression using a loess function. The shaded region represents a 95% confidence interval.

Data are represented as mean ± SEM (C – E). Each dot represents individual technical replicates (A, B) or the average of two technical replicates from each of 3 independent passages (C, E). Prevalence (G) or mean relative abundance (H) represented. Statistics by t-test (A, B) or two-way ANOVA with Tukey’s multiple comparisons test (C, E). ns (not significant) p >0.05, *p <0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Rates of early-life C. difficile colonization are associated with industrialization

Studies of C. difficile prevalence in infants have disproportionally sampled cohorts from industrialized communities (12). We therefore conducted a geographic comparative analysis of C. difficile prevalence in infants from industrialized, transitional, and non-industrialized communities, as defined by the United Nations Statistics Division (Fig. 5F) (39–50). While sequencing bias in our dataset favors samples from infants born in industrialized communities, our findings reveal a disparity in both C. difficile prevalence and relative abundance between the three settings (Fig. 5G, H). Specifically, C. difficile is significantly more prevalent and abundant in industrialized communities compared to non-industrialized communities, while transitional communities harbor an intermediate level of prevalence (Fig. 5G, H). These results suggest that early-life responses to C. difficile observed here may not reflect typical intestinal development in populations with lower exposure to this pathogen. C. difficile colonization may therefore represent an emerging mechanism by which industrialization shapes epithelial-immune development and health trajectories.

Discussion

Early-life colonization with C. difficile has long been considered an inconsequential feature of the infant microbiome, given that infants harbor toxigenic strains without developing clinical disease. Our findings fundamentally challenge this dogma. We demonstrate that even in the absence of clinical disease, colonization with toxigenic C. difficile reshapes intestinal stem cell function in neonatal mice and human infants, resulting in persistent alterations to the intestinal epithelium and immune system. Moreover, C. difficile colonization was most prevalent among infants from industrialized communities, raising the possibility that exposure to this pathogen represents an underappreciated environmental factor influencing host development (4). Several important questions remain, including the mechanisms by which infants colonized with C. difficile avoid clinical pathology despite toxin-mediated epithelial reprogramming. Additionally, the extent to which C. difficile-mediated perturbations to intestinal epithelial and immune development predispose to inflammatory or metabolic disease, or alternatively shape host development in beneficial ways, warrants further investigation. Finally, the extent to which timing of colonization, its duration, and strain-level variation in toxin production modulate these effects also remains to be determined.

Together, these findings reframe how asymptomatic C. difficile carriage in infancy should be considered clinically. If subclinical colonization during a critical developmental window has lasting consequences for epithelial and immune development, then the current practice of treating infant carriage as benign may warrant reassessment. More broadly, this study supports an emerging paradigm in which early-life colonization by toxin-producing pathogens shapes neonatal development and long-term health trajectories.

Supplementary Material

Semon-Manzer.Supplementary Materials.pdf
ady2886.Semon-Manzer.etal.Supp Table 2.csv
ady2886.Semon-Manzer.etal.Supp Table 8.xlsx
ady2886.Semon-Manzer.etal.Supp Table 1.csv
ady2886.Semon-Manzer.etal.Supp Table 7.xlsx
ady2886.Semon-Manzer.etal.Supp Table 3.csv

Materials and Methods

Figs. S1 to S10

Tables S1–S8

References (52–87)

Acknowledgments:

We thank the patients and families involved in the Infant Growth and Microbiome (IGRAM) Study, the Microbiome, the Antibiotics, Growth Infant Cohort (MAGIC), and all additional studies associated with this work for their participation. We also thank the teams involved in these studies for their support. We thank the technical cores at CHOP including the Flow Cytometry Core and Dr. Florin Tuluc and Jen Murray for their technical support in flow cytometry experiments, and the Center for Applied Genomics and Jonathan Billings for support with bulk RNA-seq of IECs. We thank the CHOP Gastrointestinal Epithelium Modeling Program (RRID: SCR_026402) for human biopsies, organoids, and protocols. We also thank the Penn Vet Comparative Pathology Core including Dr. Enrico Radaelli and Esha Banerjee with the Penn Vet Comparative Pathology Core for their contribution with immunofluorescent staining of intestinal tissues. We thank the Zackular laboratory as well as Dr. Ken Cadwell and the Cadwell laboratory at the University of Pennsylvania for thoughtful discussions and critical feedback that helped guide this work. All materials used in this manuscript are available from the authors upon request.

Funding:

National Institutes of Health grant R35GM138369 (JPZ)

Center for Microbial Medicine at the Children’s Hospital of Philadelphia (JPZ)

National Institutes of Health grant 5T32AI141393 (AS)

National Institutes of Health grant R01DK107565 (BSZ, GDW)

National Institutes of Health grant R37AI095755 (DBL)

National Institutes of Health grant R01AI158830 (MCA)

H-MARC Core for the Center for Molecular Studies in Digestive and Liver Diseases P30DK050306 (GDW)

Penn Center for Nutritional Science and Medicine (GDW)

National Institutes of Health grant R01DK133453 (MAS)

PennCHOP Microbiome Program (CET, KB, GDW)

Howard Hughes Medical Institute Hanna H. Gray finalist award (HSM)

Burroughs Wellcome Fund Investigators in the Pathogenesis of Infectious Disease Award 1356736 (JPZ)

Burroughs Wellcome Fund PDEP Award 1463880 (HSM)

CHOP SPAR Fellowship (HSM)

CHOP Bridge to Faculty Program (HSM)

Chappell Culpeper Family Foundation Fellowship (HSM, RCG, CTi, QS)

Abramson Cancer Center Support Grant (NIH P30 CA016520)

NIH Shared Instrumentation Grant (S10 OD023465–01A1)

Penn Center for Molecular Studies in Digestive and Liver Diseases (NIH P30 DK050306)

Penn Vet IIZD Core pilot grant opportunity 2022

Donation from the American Beverage Foundation for a Healthy America to the Children’s Hospital of Philadelphia to support the Healthy Weight Program.

NIH National Center for Research Resources Clinical and Translational Science Program (UL1TR001878)

Footnotes

Competing interests: In accordance with the University of Pennsylvania and Children’s Hospital of Philadelphia policies and procedures and our ethical obligations as researchers, we report that D.W. is named on patents that describe the use of nucleoside-modified mRNA as a platform to deliver therapeutic proteins and vaccines. D.W. and M.-G.A. are named on patents describing the use of lipids nanoparticles, and lipid compositions for nucleic acid delivery and vaccination. We have disclosed those interests fully to the University of Pennsylvania and Children’s Hospital of Philadelphia and have in place an approved plan for managing any potential conflicts arising from licensing of our patents. The University of Pennsylvania and the Children’s Hospital of Philadelphia submitted a patent application covering C. difficile vaccines detailed in this study. J.P.Z. has consulted for Vedanta Biosciences, Inc. and AstraZeneca. M.-G.A. serves as a scientific adviser for AfriGen Biologics, and also has an ownership stake in RNA Technologies. D.W. serves as a scientific advisor for Arcturus Therapeutics, Cabaletta Bio, and Versatope Therapeutics, and also has ownership stakes in Capstan Therapeutics, Orbital Therapeutics, Zipcode Bio, and RNA Technologies. D.W. receives royalties from CellScript and Capstan Therapeutics. All other authors declare no conflicts of interest.

Data and materials availability:

  • All materials used in this manuscript are available from the authors upon request.

  • Data tables are available through Dryad: DOI: 10.5061/dryad.ns1rn8q78(51).

  • C. difficile whole genome sequencing data from infants and children generated from this study are publicly available at the NCBI bio-project repository: PRJNA1246172 and PRJNA1246176. C. difficile sequences from children and adult were sourced from previously published datasets(20–23) and are available at the NCBI bio-project repository: PRJNA340238, PRJNA659471, PRJNA821832, PRJNA821830, and PRJNA524299. The raw sequencing reads and genome assemblies of two C. difficile samples isolated from stool of infants born at CHOP are available at NCBI bio-project repository PRJNA1445232. The accession numbers are provided in Supplementary Table 7.

  • Microbiome sequencing data generated from this study are publicly available at the NCBI bio-project repository: PRJNA1240810.

  • Human microbiome metagenomic sequencing data used to determine C. difficile prevalence and relative abundance were sourced from publicly available datasets that were previously published(39–49) and available at the NCBI bio-project repository: PRJEB49206, PRJEB51728, PRJEB6456, PRJNA268964, PRJNA290380, PRJNA504891, PRJNA549787, PRJNA1145027, PRJNA1106565, PRJNA1042647, PRJNA1173239.

  • RNA-seq raw sequence data generated from this study are available at the NCBI bio-project repository: GSE293816. RNA-seq raw sequence data from adult mouse intestinal epithelial cells during CDI were sourced from previously published datasets(30) that are available at the NCBI bio-project repository: PRJNA995763.

References and Notes:

  • 1.Arrieta MC, Stiemsma LT, Amenyogbe N, Brown EM, Finlay B, The intestinal microbiome in early life: health and disease. Front Immunol 5, 427 (2014). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Al Nabhani Z et al. , A Weaning Reaction to Microbiota Is Required for Resistance to Immunopathologies in the Adult. Immunity 50, 1276–1288 e1275 (2019). [DOI] [PubMed] [Google Scholar]
  • 3.Cahenzli J, Koller Y, Wyss M, Geuking MB, McCoy KD, Intestinal microbial diversity during early-life colonization shapes long-term IgE levels. Cell Host Microbe 14, 559–570 (2013). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Gensollen T, Iyer SS, Kasper DL, Blumberg RS, How colonization by microbiota in early life shapes the immune system. Science 352, 539–544 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Olszak T et al. , Microbial exposure during early life has persistent effects on natural killer T cell function. Science 336, 489–493 (2012). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Collignon A et al. , Heterogeneity of Clostridium difficile isolates from infants. Eur J Pediatr 152, 319–322 (1993). [DOI] [PubMed] [Google Scholar]
  • 7.Mylonakis E, Ryan ET, Calderwood SB, Clostridium difficile--Associated diarrhea: A review. Arch Intern Med 161, 525–533 (2001). [DOI] [PubMed] [Google Scholar]
  • 8.Rousseau C et al. , Clostridium difficile carriage in healthy infants in the community: a potential reservoir for pathogenic strains. Clin Infect Dis 55, 1209–1215 (2012). [DOI] [PubMed] [Google Scholar]
  • 9.Kelly CP, LaMont JT, Clostridium difficile infection. Annu Rev Med 49, 375–390 (1998). [DOI] [PubMed] [Google Scholar]
  • 10.Lessa FC, Gould CV, McDonald LC, Current status of Clostridium difficile infection epidemiology. Clin Infect Dis 55 Suppl 2, S65–70 (2012). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Lessa FC et al. , Burden of Clostridium difficile infection in the United States. N Engl J Med 372, 825–834 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Jangi S, Lamont JT, Asymptomatic colonization by Clostridium difficile in infants: implications for disease in later life. J Pediatr Gastroenterol Nutr 51, 2–7 (2010). [DOI] [PubMed] [Google Scholar]
  • 13.Lees EA, Miyajima F, Pirmohamed M, Carrol ED, The role of Clostridium difficile in the paediatric and neonatal gut - a narrative review. Eur J Clin Microbiol Infect Dis 35, 1047–1057 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Schutze GE, Willoughby RE, D. Committee on Infectious, P. American Academy of, Clostridium difficile infection in infants and children. Pediatrics 131, 196–200 (2013). [DOI] [PubMed] [Google Scholar]
  • 15.Thanert R et al. , Clinical sequelae of gut microbiome development and disruption in hospitalized preterm infants. Cell Host Microbe 32, 1822–1837 e1825 (2024). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.S. R. Tougas et al. , Prevalence of Detection of Clostridioides difficile Among Asymptomatic Children: A Systematic Review and Meta-analysis. JAMA Pediatr 175, e212328 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Eglow R et al. , Diminished Clostridium difficile toxin A sensitivity in newborn rabbit ileum is associated with decreased toxin A receptor. J Clin Invest 90, 822–829 (1992). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Dallas SD, Rolfe RD, Binding of Clostridium difficile toxin A to human milk secretory component. J Med Microbiol 47, 879–888 (1998). [DOI] [PubMed] [Google Scholar]
  • 19.Rolfe RD, Binding kinetics of Clostridium difficile toxins A and B to intestinal brush border membranes from infant and adult hamsters. Infect Immun 59, 1223–1230 (1991). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Bushman FD et al. , Multi-omic Analysis of the Interaction between Clostridioides difficile Infection and Pediatric Inflammatory Bowel Disease. Cell Host Microbe 28, 422–433 e427 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Kociolek LK et al. , Clostridium difficile Whole Genome Sequencing Reveals Limited Transmission Among Symptomatic Children: A Single-Center Analysis. Clin Infect Dis 67, 229–234 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Miles-Jay A et al. , Longitudinal genomic surveillance of carriage and transmission of Clostridioides difficile in an intensive care unit. Nat Med 29, 2526–2534 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Pecora N et al. , Genomic Analysis of Clostridioides difficile in 2 Regions of the United States Reveals a Diversity of Strains and Limited Transmission. J Infect Dis 225, 121–129 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Theriot CM et al. , Cefoperazone-treated mice as an experimental platform to assess differential virulence of Clostridium difficile strains. Gut Microbes 2, 326–334 (2011). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Chen YS et al. , IL-17-producing gammadelta T cells protect against Clostridium difficile infection. J Clin Invest 130, 2377–2390 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Jose S, Madan R, Neutrophil-mediated inflammation in the pathogenesis of Clostridium difficile infections. Anaerobe 41, 85–90 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Keenan O et al. , Pharmacological reduction of neutrophil infiltration reduces Clostridioides difficile infection severity. mBio 17, e0343025 (2026). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Pickert G et al. , STAT3 links IL-22 signaling in intestinal epithelial cells to mucosal wound healing. J Exp Med 206, 1465–1472 (2009). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Mears KS et al. , Therapeutic activation of IL-22-producing innate lymphoid cells enhances host defenses to Clostridioides difficile infection. Cell Rep 44, 115438 (2025). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Tubau-Juni N et al. , Modulation of colonic immunometabolic responses during Clostridioides difficile infection ameliorates disease severity and inflammation. Sci Rep 13, 14708 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Nusse YM et al. , Parasitic helminths induce fetal-like reversion in the intestinal stem cell niche. Nature 559, 109–113 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Mustata RC et al. , Identification of Lgr5-independent spheroid-generating progenitors of the mouse fetal intestinal epithelium. Cell Rep 5, 421–432 (2013). [DOI] [PubMed] [Google Scholar]
  • 33.Barker N et al. , Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449, 1003–1007 (2007). [DOI] [PubMed] [Google Scholar]
  • 34.Kirino S et al. , Fetal reversion from diverse lineages sustains the intestinal stem cell pool and confers stress resilience. Commun Biol 9, 255 (2026). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Clevers H, Shifting paradigms in tissue stem cell biology: Insights from the intestine. Cell 189, 706–724 (2026). [DOI] [PubMed] [Google Scholar]
  • 36.Mileto SJ et al. , Clostridioides difficile infection damages colonic stem cells via TcdB, impairing epithelial repair and recovery from disease. Proc Natl Acad Sci U S A 117, 8064–8073 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Le Monnier A et al. , Clostridioides difficile Immunity During Pregnancy and Passive Antibody Transfer to Neonates from Cord Blood and Breast Milk. Toxins (Basel) 18, (2026). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Alameh MG et al. , A multivalent mRNA-LNP vaccine protects against Clostridioides difficile infection. Science 386, 69–75 (2024). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Benitez AJ et al. , Antibiotic exposure is associated with minimal gut microbiome perturbations in healthy term infants. Microbiome 13, 21 (2025). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.D'Souza AW et al. , Cotrimoxazole Prophylaxis Increases Resistance Gene Prevalence and alpha-Diversity but Decreases beta-Diversity in the Gut Microbiome of Human Immunodeficiency Virus-Exposed, Uninfected Infants. Clin Infect Dis 71, 2858–2868 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Fragiadakis GK et al. , Links between environment, diet, and the hunter-gatherer microbiome. Gut Microbes 10, 216–227 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Jha AR et al. , Gut microbiome transition across a lifestyle gradient in Himalaya. PLoS Biol 16, e2005396 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Pasolli E et al. , Extensive Unexplored Human Microbiome Diversity Revealed by Over 150,000 Genomes from Metagenomes Spanning Age, Geography, and Lifestyle. Cell 176, 649–662 e620 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Pehrsson EC et al. , Interconnected microbiomes and resistomes in low-income human habitats. Nature 533, 212–216 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Rimal B et al. , Bile salt hydrolase catalyses formation of amine-conjugated bile acids. Nature 626, 859–863 (2024). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Robertson RC et al. , The gut microbiome and early-life growth in a population with high prevalence of stunting. Nat Commun 14, 654 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Smits SA et al. , Seasonal cycling in the gut microbiome of the Hadza hunter-gatherers of Tanzania. Science 357, 802–806 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Soto Ocana J et al. , Metal availability shapes early life microbial ecology and community succession. mBio 15, e0153424 (2024). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Tisza MJ, Buck CB, A catalog of tens of thousands of viruses from human metagenomes reveals hidden associations with chronic diseases. Proc Natl Acad Sci U S A 118, (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.“World Economic Situation and Prospects 2025,” (United Nations, Department of Economic and Social Affairs, 2025). [Google Scholar]
  • 51.Semon AM, Haider Keenan, Orlaith et al. (Dryad, 2026).
  • 52.Perez J, Springthorpe VS, Sattar SA, Clospore: a liquid medium for producing high titers of semi-purified spores of Clostridium difficile. J AOAC Int 94, 618–626 (2011). [PubMed] [Google Scholar]
  • 53.Karakasheva TA et al. , Patient-derived Colonoids From Disease-spared Tissue Retain Inflammatory Bowel Disease-specific Transcriptomic Signatures. Gastro Hep Adv 2, 830–842 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Fujii M et al. , Human Intestinal Organoids Maintain Self-Renewal Capacity and Cellular Diversity in Niche-Inspired Culture Condition. Cell Stem Cell 23, 787–793 e786 (2018). [DOI] [PubMed] [Google Scholar]
  • 55.Miyoshi H, Stappenbeck TS, In vitro expansion and genetic modification of gastrointestinal stem cells in spheroid culture. Nat Protoc 8, 2471–2482 (2013). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 56.VanDussen KL et al. , Guiding Principles: Reporting Elements for Gastrointestinal Organoid Research. Cell Mol Gastroenterol Hepatol, 101772 (2026). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 57.Schwengers O et al. , Bakta: rapid and standardized annotation of bacterial genomes via alignment-free sequence identification. Microb Genom 7, 000685 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 58.Tonkin-Hill G et al. , Producing polished prokaryotic pangenomes with the Panaroo pipeline. Genome Biol 21, 180 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 59.Minh BQ et al. , IQ-TREE 2: New Models and Efficient Methods for Phylogenetic Inference in the Genomic Era. Mol Biol Evol 37, 1530–1534 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 60.Lanave C, Preparata G, Saccone C, Serio G, A new method for calculating evolutionary substitution rates. J Mol Evol 20, 86–93 (1984). [DOI] [PubMed] [Google Scholar]
  • 61.Hoang DT, Chernomor O, von Haeseler A, Minh BQ, Vinh LS, UFBoot2: Improving the Ultrafast Bootstrap Approximation. Mol Biol Evol 35, 518–522 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 62.Yu G, Smith DK, Zhu H, Guan Y, Lam TT-Y, ggtree: an r package for visualization and annotation of phylogenetic trees with their covariates and other associated data. Methods in Ecology and Evolution 8, 28–36 (2016). [Google Scholar]
  • 63.Dingle KE et al. , Evolutionary history of the Clostridium difficile pathogenicity locus. Genome Biol Evol 6, 36–52 (2014). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 64.Braun V, Hundsberger T, Leukel P, Sauerborn M, von Eichel-Streiber C, Definition of the single integration site of the pathogenicity locus in Clostridium difficile. Gene 181, 29–38 (1996). [DOI] [PubMed] [Google Scholar]
  • 65.Clarke EL et al. , Sunbeam: an extensible pipeline for analyzing metagenomic sequencing experiments. Microbiome 7, 46 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 66.McMurdie PJ, Holmes S, phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data. PLoS One 8, e61217 (2013). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 67.Olm MR et al. , Robust variation in infant gut microbiome assembly across a spectrum of lifestyles. Science 376, 1220–1223 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 68.Marvin SA et al. , Type I Interferon Response Limits Astrovirus Replication and Protects against Increased Barrier Permeability In Vitro and In Vivo. J Virol 90, 1988–1996 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 69.Yokoyama CC et al. , Adaptive immunity restricts replication of novel murine astroviruses. J Virol 86, 12262–12270 (2012). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 70.Frey A, Di Canzio J, Zurakowski D, A statistically defined endpoint titer determination method for immunoassays. J Immunol Methods 221, 35–41 (1998). [DOI] [PubMed] [Google Scholar]
  • 71.Andrews S. FastQC: A Quality Control Tool for High Throughput Sequence Data. https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ (2010).
  • 72.Ewels P, Magnusson M, Lundin S, Kaller M, MultiQC: summarize analysis results for multiple tools and samples in a single report. Bioinformatics 32, 3047–3048 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 73.Bolger AM, Lohse M, Usadel B, Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 74.OĽeary NA et al. , Exploring and retrieving sequence and metadata for species across the tree of life with NCBI Datasets. Sci Data 11, 732 (2024). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 75.Dobin A et al. , STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 76.Li H et al. , The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 77.Anders S, Pyl PT, Huber W, HTSeq--a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166–169 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 78.Robinson MD, McCarthy DJ, Smyth GK, edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 79.Mootha VK et al. , PGC-1alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nat Genet 34, 267–273 (2003). [DOI] [PubMed] [Google Scholar]
  • 80.Subramanian A et al. , Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A 102, 15545–15550 (2005). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 81.Liberzon A et al. , The Molecular Signatures Database (MSigDB) hallmark gene set collection. Cell Syst 1, 417–425 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 82.Castanza AS et al. , Extending support for mouse data in the Molecular Signatures Database (MSigDB). Nat Methods 20, 1619–1620 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 83.Wickham H, ggplot2. Wiley interdisciplinary reviews: computational statistics 3, 180–185 (2011). [Google Scholar]
  • 84.Conway JR, Lex A, Gehlenborg N, UpSetR: an R package for the visualization of intersecting sets and their properties. Bioinformatics 33, 2938–2940 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 85.Radaelli E et al. , Mitochondrial defects caused by PARL deficiency lead to arrested spermatogenesis and ferroptosis. Elife 12, (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 86.Bankhead P et al. , QuPath: Open source software for digital pathology image analysis. Sci Rep 7, 16878 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 87.Leonardi I et al. , Mucosal fungi promote gut barrier function and social behavior via Type 17 immunity. Cell 185, 831–846 e814 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Semon-Manzer.Supplementary Materials.pdf
NIHMS2206797-supplement-Semon-Manzer_Supplementary_Materials_pdf.pdf (8.4MB, pdf)
ady2886.Semon-Manzer.etal.Supp Table 2.csv
NIHMS2206797-supplement-ady2886_Semon-Manzer_etal_Supp_Table_2_csv.csv (1.5KB, csv)
ady2886.Semon-Manzer.etal.Supp Table 8.xlsx
NIHMS2206797-supplement-ady2886_Semon-Manzer_etal_Supp_Table_8_xlsx.xlsx (93.6KB, xlsx)
ady2886.Semon-Manzer.etal.Supp Table 1.csv
NIHMS2206797-supplement-ady2886_Semon-Manzer_etal_Supp_Table_1_csv.csv (411B, csv)
ady2886.Semon-Manzer.etal.Supp Table 7.xlsx
NIHMS2206797-supplement-ady2886_Semon-Manzer_etal_Supp_Table_7_xlsx.xlsx (37.8KB, xlsx)
ady2886.Semon-Manzer.etal.Supp Table 3.csv
NIHMS2206797-supplement-ady2886_Semon-Manzer_etal_Supp_Table_3_csv.csv (5.1KB, csv)

Data Availability Statement

  • All materials used in this manuscript are available from the authors upon request.

  • Data tables are available through Dryad: DOI: 10.5061/dryad.ns1rn8q78(51).

  • C. difficile whole genome sequencing data from infants and children generated from this study are publicly available at the NCBI bio-project repository: PRJNA1246172 and PRJNA1246176. C. difficile sequences from children and adult were sourced from previously published datasets(20–23) and are available at the NCBI bio-project repository: PRJNA340238, PRJNA659471, PRJNA821832, PRJNA821830, and PRJNA524299. The raw sequencing reads and genome assemblies of two C. difficile samples isolated from stool of infants born at CHOP are available at NCBI bio-project repository PRJNA1445232. The accession numbers are provided in Supplementary Table 7.

  • Microbiome sequencing data generated from this study are publicly available at the NCBI bio-project repository: PRJNA1240810.

  • Human microbiome metagenomic sequencing data used to determine C. difficile prevalence and relative abundance were sourced from publicly available datasets that were previously published(39–49) and available at the NCBI bio-project repository: PRJEB49206, PRJEB51728, PRJEB6456, PRJNA268964, PRJNA290380, PRJNA504891, PRJNA549787, PRJNA1145027, PRJNA1106565, PRJNA1042647, PRJNA1173239.

  • RNA-seq raw sequence data generated from this study are available at the NCBI bio-project repository: GSE293816. RNA-seq raw sequence data from adult mouse intestinal epithelial cells during CDI were sourced from previously published datasets(30) that are available at the NCBI bio-project repository: PRJNA995763.

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