A High-Salt Diet Promotes Colitis by Remodeling the Gut Microbiota Toward a Virulent, Osmotolerant State

A High-Salt Diet Promotes Colitis by Remodeling the Gut Microbiota Toward a Virulent, Osmotolerant State | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A High-Salt Diet Promotes Colitis by Remodeling the Gut Microbiota Toward a Virulent, Osmotolerant State Wenjie Zhang, Yang Gao, Qiaoyu Ling, Xiaochen Zhu, Wanrong Luo, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8881149/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Background Excessive dietary salt disrupts the gut microbiota and is increasingly recognized as a contributor to the rising prevalence of chronic inflammatory diseases. In Crohn’s disease (CD), Western dietary patterns and gut microbiome dysbiosis are well‑established drivers of disease incidence. Identifying modifiable dietary factors, such as high salt intake, is therefore critical for developing improved preventive and therapeutic strategies. However, the precise effects of high-salt diets (HSDs) on the luminal electrolyte profile, the gut microbiome, and subsequent susceptibility to intestinal inflammation, however, remain poorly defined. Results Using a treatment‑naïve cohort, we show that CD patients exhibit a distinct fecal electrolyte profile characterized by elevated Na⁺ and reduced K⁺, which correlates with markers of both gut and systemic inflammation. Relative and absolute quantification further reveal increased abundance of the halophilic archaeon Halorubrum and a corresponding decrease in Methanobrevibacter . Systematic profiling of salt‑stress associated functional genes, including those involved in K⁺ transport, compatible solute transport and biosynthesis, revealed that the CD gut microbiome is enriched with salt stress response genes. A similar enrichment trend was observed in industrialized populations compared to the Hadza hunter‑gatherer communities. Phylogenetic and growth assays demonstrated that these genetic determinants are more prevalent in opportunistic pathogens, which consequently exhibit greater resistance to growth inhibition under high Na⁺ conditions. Indeed, using high‑salt selective media, we isolated salt‑tolerant opportunistic pathogens from colonic biopsies of CD patients. Through in vivo fecal microbiota transplantation, we demonstrate that a high‑Na⁺ adapted microbial community exacerbates colitis. Salt tolerance and its associated pathogenicity vary across strains. The E. coli CD09 isolate from CD patients exhibited greater salt tolerance than the model strain E. coli MG1655, and mice inoculated with CD09 developed more severe colitis. Conclusions Collectively, our findings elucidate a mechanism by which HSDs exacerbate intestinal colitis through restructuring of the gut microbial community. A high‑Na⁺ luminal environment drives microbial adaptation toward salt tolerance, amplifies virulence at both the community and single‑strain levels, and ultimately exacerbates colitis. Thus, the gut microbiota represents a promising therapeutic target for preventing and mitigating salt‑associated chronic inflammatory diseases. Western diet osmotic stress coevolution environmental adaptation Halorubrum Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction High-salt diets (HSDs) represent a significant risk factor for a spectrum of non-communicable chronic inflammatory diseases, including inflammatory bowel disease (IBD), hypertension, cardiovascular disease (CVD), and Alzheimer’s disease, and are associated with increased disease activity in multiple sclerosis 1 – 3 . A wealth of epidemiological evidence strongly supports reducing salt consumption, aligning with the World Health Organization's recommendation to limit intake to < 5 g/day 4 . Nonetheless, global consumption frequently exceeds 10 g daily, more than 70% of the sodium we eat comes from packaged, prepared and restaurant foods due to salt added for flavoring, stabilizing, preserving and reducing bacterial risk 1 . High dietary Na + elevates systemic and luminal Na + concentrations, impairing health through multiple distinct mechanisms. Initial research primarily focused on hemodynamic effects and direct pro-inflammatory immune activation, including vascular endothelial dysfunction, immune cell recruitment, and cytokine production 5 – 7 . More recent investigations have established the gut microbiota as a critical mediator of salt-sensitive pathology. The colon is a major absorptive and secretory organ for electrolytes. In healthy adults, the colon is to absorb 90% of 1.5–2 L of electrolyte-rich small intestinal fluid that enters the colon daily 8 . This flux thus represents a critical environmental stimulus for the gut microbiota. Recent studies demonstrated that a high-salt challenge depletes intestinal Lactobacillus spp., promotes the expansion of pro-inflammatory TH17 cells, lower beneficial metabolites e.g. short chain fatty acids production 9 , 10 , thereby positioning the gut microbiome as a viable therapeutic target for mitigating salt-associated disorders. Crohn’s disease (CD), a subtype of IBD, is a chronic gastrointestinal inflammation imposes a substantial symptom burden and impairs quality of life 11 . Although the precise etiology of CD remains incompletely understood, disease onset is widely accepted to involve complex interactions between environmental factors, host genetics, gut microbiota, and immune responses 12 , 13 . At the turn of the 21st century, IBD has become a global disease with accelerating incidence in newly industrialized countries in Africa, Asia, and South America, there is a pressing need for improved preventive and therapeutic strategies 14 . While the Western diet and gut microbiome dysbiosis are primary drivers of the marked rise in CD incidence in recent decades, identifying modifiable dietary risk factors such as excessive salt intake is critical for developing improved preventive and therapeutic strategies. Prior studies found that Lactobacillus mediates HSD exacerbated colitis 10 . Owing to their documented health benefits, gastrointestinal Lactobacillus species are extensively studied and commonly used as probiotics. However, Lactobacillus species constitute a minor and highly variable fraction of the colonic microbiota, representing ~ 0.01% of cultivable bacterial counts and are undetectable in nearly 25% of human fecal samples 15 . Furthermore, multi-cohort analyses in CD report inconsistent findings regarding Lactobacillus levels, with studies indicating both increases and decreases relative to healthy controls 16 , 17 . Consequently, the translational relevance of Lactobacillus -mediated, HSD-aggravated colitis to human CD pathophysiology remains unresolved. We reasoned that HSDs elevates luminal Na + concentration in the ileum and colon, thereby imposing significant salt and concomitant osmotic stress on the gut environment. Indeed, the gut archaeome in patients with colorectal cancer exhibits an enrichment of halophilic archaea compared to both adenoma patients and healthy individuals 18 . Osmolarity is a critical abiotic factor that profoundly shapes microbial ecosystems, and successful gut colonization depends on adaptation to such environmental pressures 19 , 20 . Prokaryotes employ two primary osmoregulatory strategies to thrive under salt-stressed conditions: the "salt-in" strategy, which maintains osmolarity through inorganic ion accumulation ( e.g. , K + ), and the "salt-out" strategy, which utilizes compatible solutes to balance external pressure without disrupting intracellular function 21 . While the salt tolerance of pathogens is well-characterized, the response of the broader human gut microbiome to salt-stress remains largely unknown. In this study, we analyzed a treatment naïve Crohn's disease cohort to profile fecal electrolytes (Na⁺, K⁺), quantify halophilic archaea, and assess metagenomic salt stress response genes. We characterized the growth phenotypes of representative species across a salinity gradient and investigated salt-stress adaptation at both polymicrobial community and single-strain levels, demonstrated that salt-tolerant opportunistic pathogens gain ecological advantage in the colon due to elevated luminal Na + levels. Collectively, our results provide novel mechanistic insights into how HSDs exacerbate colitis via gut microbial community restructuring, revealing potential microbiome-targeted strategies for the prevention and treatment of HSDs imposed chronic inflammation diseases. Results Fecal electrolyte analysis reveals a dysregulated luminal environment in CD The colon dynamically regulates electrolyte homeostasis through absorptive and secretory processes 22 . In CD, this balance is perturbed by the confluence of dietary habits, chronic diarrhea, mucosal inflammation, and impaired nutrient absorption 11 . We therefore hypothesized that the fecal electrolyte profile, specifically the concentrations of Na + and K + , would be altered in CD. Analysis of stool samples from CD patients (n = 108) and healthy individuals (n = 124) confirmed this hypothesis. CD patients exhibited elevated Na + , reduced K + , and a consequently elevated Na + /K + ratio (Fig. 1 A-C). Fecal electrolyte profiles are significantly associated with CD phenotype and severity. Stratification of patients by disease behavior revealed that fecal K⁺ levels were approximately 1.5-fold higher in the non-stricturing, non-penetrating (B1) phenotype than in the stricturing (B2) or penetrating (B3) phenotypes (Figure S1 A). The fecal Na⁺/K⁺ ratio correlated positively with the Crohn's Disease Activity Index (CDAI), the gut inflammation marker fecal calprotectin, and systemic inflammation markers (neutrophil% and mean platelet volume), and correlated negatively with platelet count (Figure S1 B). In receiver operating characteristic (ROC) analysis, both fecal K + (AUC = 0.87) and the Na + /K + ratio (AUC = 0.79) demonstrated significantly greater discriminatory power for CD than platelet count (PLT, AUC = 0.69) (Fig. 1 D). (A-C) Fecal analysis reveals significantly elevated Na⁺ (A), reduced K⁺ (B), and a consequently higher Na⁺/K⁺ ratio (C) in CD individuals compared to HCs. The internal line represents the median. Significance was determined by non-parametric Mann–Whitney test. (D) Diagnostic performance of fecal Na⁺ and K⁺ levels for discriminating CD. CRP, C reactive protein; PLT, platelet count. Dietary salt and impaired transport drive intestinal electrolyte dysregulation Given the elevated fecal Na + and reduced K + levels observed in CD patients, we next investigated whether an HSD drives this luminal electrolyte imbalance. To test this, mice were fed an HSD for 4 weeks, after which luminal Na⁺ and K⁺ concentrations were quantified along the intestinal tract (Fig. 2 A). While control mice maintained consistent Na⁺, K⁺, and Na⁺/K⁺ ratios from the small intestine to the colon, HSD feeding significantly perturbed this homeostasis (Figure S2 A-C). Specifically, HSD led to elevated Na⁺ in the distal small intestine and cecum, a progressive decrease in K⁺ from the proximal small intestine to the colon, and a corresponding increase in the Na⁺/K⁺ ratio along the colonic axis compared to mice on NSD (Fig. 2 B). However, the colonic content of HSD-fed mice did not fully recapitulate the human CD phenotype, which features high Na⁺ and low K⁺ (Fig. 1 A-C). We reasoned that this discrepancy stems from the preserved electrolyte absorption capacity in healthy murine colon, a function known to be impaired in CD patients 23 . We analyzed the expression of key Na + transporters, NHE1–3, ENaC, and Na⁺/K⁺-ATPase, in intestinal biopsies from newly diagnosed CD patients (n = 46) and non-disease controls (n = 44) from the FAH-SYSU cohort 17 . Our analysis revealed a significant downregulation of NHE1 and NHE2 in inflamed CD mucosa, with a strong decreasing trend for Na⁺/K⁺-ATPase. This pattern of downregulation was corroborated in three independent IBD cohorts (GSE83687 24 , E-MTAB-5464 25 , HMP IBD 26 ) (Fig. 2 C, D). We therefore hypothesized that the CD luminal Na + /K + profile results from the synergy of high dietary salt intake and impaired host electrolyte transport. To test this, we administered the Na + transport inhibitors amiloride and cariporide to mice 27 . Cariporide is a potent and selective NHE1 inhibitor, whereas amiloride is less specific, also inhibiting other NHE isoforms and ENaC. Consistent with our hypothesis, mice treated with amiloride exhibited an elevated fecal Na⁺/K⁺ ratio, driven by increased Na⁺ and decreased K⁺ concentrations (Fig. 2 E, F; Figure S2 D-G). This result directly validates that combined high dietary salt and impaired electrolyte transport synergistically generate a luminal environment of high Na + and low K + of CD. (A, B) Schematic diagram showing the HSD challenge experimental design and sampling timeline (A). A HSD altered intestinal Na⁺ and K⁺ concentrations (B). Mean ± SD. Significance was determined by unpaired t test. (C, D) Colonic Na + transporters (C). Analysis of mucosal transcriptomic data from multiple IBD cohorts demonstrates significant downregulation of key Na + transporter genes in CD patients compared to various control tissues (D). FAH-SYSU: non-disease; IBDMBD: symptomatic non-IBD; E-MTAB5464: non-disease; GSE83687: normal tissue from cancer patients. The internal line represents the median. Significance was determined by non-parametric Mann–Whitney test. (E, F) Schematic diagram showing the experimental design and sampling timeline (E). Pharmacological inhibition of Na + transport in mice elevates luminal Na⁺ and reduces K⁺ (F). Mean ± SD. Significance was determined by unpaired t test. CD fecal samples show increased abundance of halophilic archaeon Halorubrum While the human archaeome remains largely uncharacterized, previous studies indicate the gut harbors a diverse archaeal community beyond the well-studied Methanobrevibacter 28 , with halophilic archaea reportedly enriched in colorectal cancer 18 . Indeed, Halorubrum lipolyticum originally isolated from hypersaline environments like salt lakes, it is now recognized as a component of the diverse human gut archaeome 28 . We therefore sought to characterize the archaeal composition in CD patients compared to healthy subjects. Using a conservative, archaea-specific 16S rRNA gene amplification and a bioinformatic pipeline, we profiled the fecal archaeome. Results revealed a diverse community dominated by the genera Methanobrevibacter and a halophilic Archaea Halorubrum (Fig. 3 A). Fecal samples from CD individuals (n = 51) had significant enrichment of Halorubrum and depletion of methanogenic archaea Methanobrevibacter than HCs (n = 131) (Fig. 3 B). To validate these findings, we developed a multiplex droplet digital PCR (ddPCR) assay targeting both genera 29 . The ddPCR results confirmed a reduction in the absolute abundance of Methanobrevibacter and a concomitant increase in Halorubrum in CD (n = 50) than HCs (n = 126), supporting the conclusion that a Na + -enriched colonic environment selectively enriches for halophilic archaea (Fig. 3 C). (A) Archaeal community profiles in CD patients and healthy controls. (B, C) CD individuals demonstrate elevated relative (B) and absolute (C) abundance of Halorubrum and reduced Methanobrevebacter . Significance was determined by non-parametric Mann–Whitney test. A Na-enriched colon niche selects for a salt-tolerant microbiota The initial phase of bacterial salt stress adaptation often involves the cytoplasmic accumulation of K⁺ ions, which serve as inorganic osmoprotectants to rapidly counterbalance external osmotic pressure 30 , 31 . A subsequent, secondary response entails the biosynthesis and/or uptake of compatible solutes, such as carnitine, glycine betaine, trahelose, ectoine, proline etc , which act as organic osmoprotectants to restore cell volume and turgor pressure 32 . To systematically investigate this adaptive machinery, we compiled a comprehensive set of genes (clusters) (29) encoding: 1) transporters for active K⁺ uptake (5), 2) transporters for compatible solutes (20), and 3) enzymes for compatible solute biosynthesis (4) (Fig. 4 A, Supplementary Dataset 1). We conducted a comprehensive investigation into the salt-stress tolerance of the human gut microbiome, focusing on key genes responsible for active K + transport, compatible solute transport and biosynthesis. Our analysis was based on stool metagenomic samples from two independent IBD cohorts, FAH-SYSU and PRISM. We found that the abundances of gene (clusters) involved in salt adaptation, including K⁺ transporters ( kdpFABC , kefBG , kefCF ), compatible solute transporters ( opuE / putP , betaine-choline-carnitine ABC transporters, and bcct family members), and the betaine biosynthesis cluster ( betAB ), were significantly enriched in the CD metagenome (Fig. 4 B). This increasing trend was consistently observed in the independent PRISM cohort, though statistical significance was not reached for some gene clusters. We reasoned that the dramatic rise in dietary Na + intake following industrialization drives gut microbiota to a salt-tolerant structure. To test the hypothesis, we examined gut metagenomes from populations representing a spectrum of industrialization, including the Hadza hunter‑gatherers of Tanzania (n = 125) and comparative populations from Nepal and California (n = 54) 33 . Consistent with findings from FAH-SYSU and PRISM cohorts, several genes encoding K⁺ transporters ( kdpFABC ) and compatible solute transporters ( bcc ABC transporter ) reached statistical significance in the industralised populations (Nepal and California) comparing to non-industralised population (Hadza) (Figure S3), confirming the enrichment of salt‑stress response genes in industrialized populations. Phylogenetic distribution of salt tolerant human associated bacteria To systematically evaluate the phylogenetic distribution of salinity-responsive genes in the human microbiome, we profiled their presence across 1,640 reference genomes from the Human Microbiome Project (HMP). This analysis revealed a significant enrichment of salt-stress response genes in opportunistic pathogens, particularly facultative and obligate aerobes from Pseudomonadota and Bacillota phyla, such as E. coli , Proteus mirabilis , Streptococcus pneumoniae , and Staphylococcus aureus (Fig. 5 A, Supplementary Dataset 2). Skin is a high osmotic stress environment for bacteria due to low moisture and high salt concentrations from evaporated sweat. As expected, salt-stress tolerant bacteria were also observed in skin-associated members of the Actinomycetota phylum, including Cutibacterium acnes and Corynebacterium species (Fig. 5 A, Supplementary Dataset 2). Notably, 17.5% of genomes (287/1,640) harbored ≥ 4 salt-tolerance genes (Figure S4A). These genotypes were predominantly from the Pseudomonadota phylum and the Staphylococcaceae and Enterococcaceae families from the Bacillota phylum, taxa commonly enriched in the CD gut microbiome 26 , 34 . Conversely, bacterial families strongly associated with host health state, including Lachnospiraceae and Oscillospiraceae ( Bacillota ), Prevotellaceae and Rikenellaceae ( Bacteroidota ), and Akkermansiaceae ( Verrucomicrobiota ) 35 , typically harbored < 4 salt-stress response genes (Fig. 5 A). Isolation of salt-tolerant bacteria from CD mucosal biopsy Given the observed enrichment of salt-stress response genes in the CD metagenome, we next sought to determine whether viable salt-tolerant bacteria colonise the CD intestinal mucosa. Mucosal biopsies from 6 CD patients were homogenized and plated on rich medium supplemented with 6% NaCl, a concentration that inhibits most bacteria but selectively permits the growth of halotolerant species, as established in standard salinity stress assays ( e.g. , 6.5% NaCl broth for Staphylococusi ). Consistent with our genomic predictions, we successfully isolated 9 halotolerant species, including 7 from the Pseudomonadota phylum and 2 from the Bacillota phylum (Fig. 5 B). Salinity stress provides an ecological advantage to salt tolerant bacteria Bacteria respond to salt stress differently, with salt tolerant strains outcompete salt sensitive strains to gain ecological advantage in salt stressed environment. We hypothesized that strains harboring multiple salt-stress response genes would exhibit enhanced salt tolerance, manifesting as reduced growth inhibition under high Na + conditions. To test this, we cultured a panel of gut bacteria in media containing 0.5%, 1.5%, or 2.5% NaCl. The highest concentration was selected to approximate the mean fecal Na + concentration measured in individuals within the 4th quartile of CD patients. This concentration corresponds to 21.89 Na⁺ mg/g (dry stool) (Figure S5), equivalent to a 2.5% NaCl solution assuming 85% fecal water content. Consistent with our hypothesis, elevated Na + concentration progressively inhibited the growth of various commensal gastrointestinal bacteria. The growth of commensal bacteria, Bacteroides ovatus , Clostridium butyricum, Bacteroides thetaiotaomicron , Akkermansia muciniphila , Clostridium sporogenes , and probiotic Bifidobacterium longum were significantly inhibited by increasing NaCl in the medium, with B. ovatus , and A. muciniphila was completely inhibited at 1.5% and 2.5% NaCl. In contrast, E. coli , S. aureus , and P. mirabilis , all of which carrying > = 4 salt-tolerance genes, maintained growth rates at 2.5% NaCl that were comparable to those observed at 0.5% (Fig. 5 C, D, Supplementary Dataset 3). The salt-stress adapted microbiota exacerbates intestinal inflammation We proposed that excess dietary salt leads to elevated gut luminal Na + levels, which promote salt tolerant microbial communities which worsen colon inflammation. To test this, mice received a high salt, high protein diet (HSHP), modeling a dietary pattern associated with CD 36 . After 21 days, these mice showed a thinner colon mucus layer and lower muc2 gene expression compared to mice on an isocaloric normal salt high protein diet (NSHP) (Fig. 6 A-D). Their gut microbiomes also formed distinct clusters (Fig. 6 E), exhibited reduced Shannon diversity (Fig. 6 F), and showed an increasing trend in the abundance of salt-stress response genes. Among these, kdpA , kdpC , and kimA reached statistical significance (Fig. 6 G). To test whether a salt-stress adapted microbiota is sufficient to exacerbate inflammation, we performed fecal transplants. Donor mice were fed either a HSHP or NSHP. Fecal microbiota transplantation (FMT) experiments were performed using donors from each dietary group at 10 and 21 days’ post-diet initiation. After inducing colitis, mice that received fecal transplant from HSHP donors consistently developed significantly shorter colons, indicating more severe inflammation (Fig. 6 H-J). Notably, this effect required the combined high salt and high protein diet, as transplants from mice on a high salt normal chow diet post 10 and 28 days did not cause shorter colon length (Figure S6A-C). This demonstrates that a diet rich in both salt and protein shapes a pro-inflammatory gut microbiome capable of driving more severe colitis. (A) Experimental design, timeline, and sampling strategy for donor mice. (B-D) Donor mice fed a high-salt, high-protein (HSHP) diet exhibit a thinner colonic mucus layer (B, C) and reduced muc2 gene expression (D) compared to mice on a normal-salt, high-protein (NSHP) diet. Mean ± SD. Significance was determined by unpaired t-test. (E-G) The gut microbiota of HSHP-fed donors forms a distinct cluster (E), shows reduced Shannon diversity (F), and exhibits enriched microbial salt-stress gene abundance (G). Dot size represents gene prevalence and color indicates mean gene abundance (RPKM). Key genes are highlighted by function (K⁺ transport, osmolyte metabolism). G⁻, Gram-negative; G⁺, Gram-positive. bcc ABC transporter , betaine-choline-carnitine ABC transporter . (H-J) Experimental design for fecal microbiota transplantation (FMT), showing timeline and sampling at two time points (days 10 and 21) from HSHP-fed donors (H). Following FMT and DSS challenge, representative colon morphology (I), and colon length measurements (J) of recipient mice at endpoint. Mean ± SD. Significance was determined by unpaired t-test. Salt tolerance in E. coli associates with pathogenicity in colitis Bacterial salt tolerance varied between strains. We compared the halotolerance of E. coli CD09, a clinical isolate from this study, and the laboratory strain E. coli MG1655 by culturing them in K + -depleted M9 medium with 0.5% or 2.5% NaCl. While both strains exhibited increased doubling times at 2.5% NaCl compared to 0.5%, E. coli CD09 displayed significantly greater salt tolerance, indicated by significantly shorter doubling time at 2.5% NaCl (Fig. 7 A, B). Despite sharing a core set of salt-stress response genes with the laboratory strain E. coli MG1655 (Supplementary Dataset 3), the clinical isolate E. coli CD09 exhibited significantly greater salt tolerance, indicating additional, uncharacterized genetic or regulatory mechanisms that likely contribute to its superior salt stress resistance. Previous studies demonstrated that bacteria salt tolerance is associated with infection, biofilm formation, antibiotic resistance and virulence etc 37 , hence we reasoned that enhanced salt tolerance of E.coli CD09 was associated with increased pathogenicity in vivo . Abx-treated mice were colonized with either E. coli CD09 or MG1655 and then subjected to DSS-induced colitis (Fig. 7 C). Mice colonized with the E. coli CD09 isolate developed more severe disease, as indicated by significantly greater body weight loss, higher disease activity index scores, and exacerbated histopathological damage, including extensive mucosal erosion, crypt loss, and inflammatory cell infiltration (Fig. 7 D-I). (A, B) E. coli CD9 exhibited enhanced growth under high Na⁺ stress as shown by growth curves (A) and doubling time (B). Mean ± SEM. Statistical significance was determined by one-way ANOVA with multiple comparisons for panel (B). (C-I) Schematic diagram showing the experimental design, timeline and sampling strategy (C). Body weight (D) and disease activity index (E) were monitored following DSS administration. Colonic morphologies (F), colon length (G), representative H&E-stained colon sections (H) and histological assessment (I) at the termination of the experiment on day 8. Mean ± SD. Statistical significance was determined by unpaired t-test for panel G and I. Discussion While elevated Na + levels directly impact immune responses, they also drive the gut microbial community toward a more virulent structure, thus having synergistic effects, leading to more severe disease. The composition and function of the gut microbiota are fundamentally shaped by the physicochemical environment, wherein each taxonomic unit proliferates only when local conditions meet its specific biochemical and metabolic requirements. While microbial salinity adaptation has been extensively investigated in environments of marine, estuary and salt lakes, the role of gut luminal salinity driven by Na + and K + concentrations has been largely overlooked. While previous work has centered on the salt-induced depletion of Lactobacillus in the gut, this study provides a more comprehensive view of microbiome restructuring. To better understand how to remediate diseases associated with both dysbiosis and environmental perturbation of the gut, it is crucial to establish the physical parameter ranges in healthy and perturbed environments, including the microenvironments along the intestine that are relevant to disease states. In CD, concurrent excessive salt intake and impaired Na + absorption generate a high Na + , low K + luminal environment that imposes significant salt stress on gut microbiota. Here, we characterized the salt-stress adaptation from three aspects: 1) halophilic archaeon Halorubrum is enriched in CD patients indicated by both relative and absolute abundance quantification. Halorubrum was detected by archaea specific and Halorubrum specific primers, although it has not been cultured; 2) the enrichment of bacterial salt-stress response genes by metagenomic analysis; 3) isolation and culturing of halotolerant bacteria from CD biopsies. In summary, we demonstrate that the Na + -enriched luminal environment in CD drives a compositional shift toward a more salt-tolerant microbiota. Microbial salt tolerance is associated with pathogenicity at both the microbial community and individual strain levels. Colonization of mice with a salt-stress adapted microbiota or a more halotolerant E. coli strain resulted in exacerbated colitis in vivo . A variety of strategies were employed by varying microbial linage. Opportunistic pathogens from Enterobacterales and Staphylococcaceae are enriched in CD patients 26 , 36 , which harbor multiple salt-stress response genes, hence are more sustained in high Na + medium, whereas commensal bacteria, typically enriched in HCs, such as strains from Clostridiaceae , Oscillospiraceae , and Lachnospiraceae 26 , 34 , 35 which carrying minimal salt-stress response genes, exhibit severe growth inhibition as Na + concentration rises. Salt stress is frequently linked to a broader environmental stress response, encompassing resistance to osmolarity, cold and acid shock. For instance, BCCT and ABC transporter families contribute significantly to cryotolerance 38 , 39 . Therefore, carrying salt-stress response genes enable them to survive in fluctuation environment and enhance infection 40 , 41 . Indeed, deletion of osmolyte transporters attenuated pathogenicity of L. monocytogenes 31 , 42 , 43 , Shigella 44 and Pseudomonas aeruginosa 40 . In addition, bacteria senses salinity to trigger virulence mechanism 37 . Thus, inherent environmental stress tolerance, coupled with shared genetic regulation of virulence, potentiates the pathogenicity of salt-tolerant bacteria. Our findings reveal that the gut microbiome mediates the pro-inflammatory effects of a high salt diet through ecological mechanisms that extend beyond the depletion of Lactobacillus , identifying luminal salt stress induced microbial community restructuring as a key mechanism linking HSDs to colitis development. It is noteworthy that the pro-inflammatory effect of a salt adapted microbiota displayed dietary dependence. Transplantation from HSHP donors exacerbated colitis, while microbiota from high salt, normal chow donors did not, indicating that the dietary context determines salt-driven microbial pathogenicity. Gut microbes coevolve with their host. Excessive dietary Na + is a critical global health challenge, implicated in an estimated 1.89 million deaths annually and associated with a spectrum of chronic conditions, including cardiovascular disease and IBD 45 . Epidemiological evidence consistently identifies high salt consumption, characteristic of Western diets, as a significant risk factor for CD 3 . Pre-agricultural humans are estimated to have consumed only about 768 mg of Na + per day (~ 2 g/day salt) 46 . Globally, people consume nearly double the recommended amount of Na + (5 g/day salt). The rise in Na + consumption since industrialization, largely driven by its pervasive use in processed and prepared foods, frequently results in intake that far exceeds physiological requirements. Historically, dietary salt was scarce. Consequently, non-industrialized populations such as the Hadza hunter-gatherers harbor a lower prevalence and abundance of bacterial genes that confer salt tolerance, including those for K + active transport and compatible solute transport, in comparison to industrialized populations. Their microbiomes are also enriched with taxa from the Spirochaetota phylum that carry minimal salt-stress response gene 33 . In contrast, opportunistic pathogens from the Pseudomonadota and Bacillota phyla, which typically carry > = 4 salt-stress response genes, are commonly enriched in conditions associated with excessive salt intake and Western diet, including IBD, CVD, and obesity 47 , 48 . Although we focused on CD in this study, this microbial restructuring may also contribute to other HSDs-linked diseases. In conclusion, we report that CD patients exhibit a gut microenvironment characterized by high Na + , low K + levels. This altered luminal salinity drives microbial adaptation toward salt tolerance, enhances virulence at both the community and strain levels, and ultimately exacerbates intestinal inflammation. Our findings not only establish a causal link between gut microbial stress adaptation and virulence but also highlight this adaptive pathway as a promising target for novel therapeutic interventions in CD management. Materials and Methods Human subjects All study protocols abided by the Declaration of Helsinki principles and were approved by Ethical Committees of the First Affiliated Hospital of Sun Yat-sen University. Intestinal biopsies and stool specimens were collected as part of the FAH-SYSU cohort study (2016[113], 2023[488]). Subject stool samples were collected at the FAH, SYSU gastroenterology clinic and stored at -80°C immediately. For culturing assays, tissue samples were collected and stored in cryogenic vials at -80°C until use 49 . Na + and K + quantification Intestinal luminal contents and faecal samples were lyophilized for 12 hours. Approximately 8 mg of homogenized dry sample was weighed and digested in 1 mL of 3% nitric acid (HNO₃) overnight at room temperature with gentle agitation. Na⁺ and K⁺ concentrations in the digests were quantified via flame photometry (ContrAA 800, Analytik Jena) or inductively coupled plasma optical emission spectrometry (Avio 500, PerkinElmer). Characteristic emission wavelengths of 589.9 nm for Na⁺ and 766.5 nm for K⁺ were used. All digestion and analytical steps were performed in plasticware to minimize exogenous ion contamination. Archaeome profiling and Droplet digital PCR (ddPCR) for Methanobrevibacter and Halorubrum Total microbial genomic DNA was extracted from faecal samples using the magnetic bead-based MagPure FFPE DNA/RNA Kit (Magen Biotechnology, D6364-02) and quantified spectrophotometrically (Implen GmbH, NanoPhotometer N60). Archaeal community profiling was performed following the protocol of Koskinen et al. (2017) 29 . Absolute quantification of Methanobrevibacter and Halorubrum was performed via ddPCR using the mcrA and rpoB marker genes, respectively. Gene-specific primers and probes were designed using PrimerQuest and validated by BLAST analysis. Each 20 µL ddPCR reaction contained 10 µL of ddPCR Supermix (Bio-Rad, no dUTP, Bio-Rad, 1863024), 900 nM of each primer, 250 nM of probe, and ~ 100 ng of faecal DNA. Non-template and positive controls ( M. smithii CCAM68 and H. lacusprofundi ATCC 49239) were included. Bacterial growth Bacterial strains were revived from glycerol stocks on solid media at 37°C. After 24–48 hours, single colonies were inoculated into liquid medium and grown overnight. Strains were cultured as follows: Bacteroides ovatus ATCC 8483 and Akkermansia muciniphila BAA-835 in Brain Heart Infusion (BHI) medium (HKM Bio, 024053); Bifidobacterium longum JCM11341 in BBL medium (Hope Bio, HB8777); Bacteroides thetaiotaomicron ATCC 29148, Clostridium butyricum ATCC 19398, and C. sporogenes ATCC 15579 in Chopped Meat medium (TOPBIO, MD155B) (all anaerobically). Escherichia coli MG1655, Staphylococcus aureus ATCC 29213, and Proteus mirabilis GN2 were grown aerobically in Luria broth medium (Hope Bio, HB0128). For salt-tolerance assays, overnight cultures (1:100, v/v ) were grown in medium with 0.5%, 1.5%, or 2.5% NaCl ( w/v ). Growth (OD₆₀₀) was measured at intervals in triplicate and analysed using a logistic growth model. E. coli strains MG1655 and CD09 were initially cultured in LB medium and then subcultured (1:100, v/v ) into a modified K + depleted M9 medium containing 4 mM Na₂HPO₄, 22 mM NaH₂PO₄, 18.5 mM NH₄Cl, 1 mM MgSO₄, 0.1 mM CaCl₂, 10 µg/mL thiamine, and 0.5% glucose. This defined medium was supplemented with either 0.5% or 2.5% NaCl ( w/v ) to impose osmotic stress. Cultures were grown aerobically at 37°C with shaking (250 rpm), and bacterial growth was monitored by measuring the optical density at 600 nm (OD₆₀₀). Bacteria isolation and identification Approximately 50 mg of colonic mucosal tissue from CD patients was homogenized in 500 µL PBS. The supernatant was serially diluted (10- and 100-fold) and plated on LB agar with 6% NaCl ( w/v ). After 48 hours of aerobic incubation, distinct colonies were picked and smeared onto a MALDI target plate. Spots were treated with 1.0 µL of 70% formic acid, air-dried for 5 minutes, overlaid with matrix solution (α-cyano-4-hydroxycinnamic acid saturated in 50% acetonitrile with 5% trifluoroacetic acid, w/v ), and analyzed by MALDI-TOF/MS (Bruker Biotyper smart). Spectra were collected and compared against the manufacturer’s reference for identification. Analyses of salt tolerance associated genes in Human Microbiome project (HMP) references genomes HMP references genomes (1640 genomes as of October 30, 2025) were selected and analyzed through the IMG program on the Joint Genome Institute website ( https://img.jgi.doe.gov/ ). Key genetic determinants for salt-stress adaptation, including K⁺ active transporters, osmoprotectant transporters, and biosynthetic enzymes, were systematically catalogued from the literature (Supplementary Dataset 1). Hits were manually inspected. Genomes carrying salt-stress response gene(s) were selected to generate a phylogenetic tree using phyloT ( https://phylot.biobyte.de/ ) based on NCBI taxonomy and visualized using iTOL 50 . Genome and gene IMG ID are available in Supplementary Dataset 2. Metagenomic data analysis We used ShortBRED 51 to accurately profile the abundance of genes involved in the salt-stress response in metagenomes sourced from the FAH-SYSU (BioProject: PRJNA793776) 52 , PRISM (BioProject: PRJNA400072) 53 , Hadza Hunter-Gatherers (BioProject: PRJEB49206) 33 datasets. We initially compiled a set of identified bacterial salt-stress response genes as our query sequences (Supplementary Dataset 2). ShortBRED-Identify was employed to generate markers for these key bacterial salt-stress response gene sequences using UniRef90 (June, 2025) as a reference list with an 85% cluster ID threshold. These markers were applied in ShortBRED-Quantify to assess gene abundance in paired metagenomes, which had previously undergone quality control via the KneadData workflow ( http://huttenhower.sph.harvard.edu/kneaddata ). The output from ShortBRED-Quantify was expressed as reads per kilobase per million mapped reads (RPKM). Animal Studies Male SPF C57BL/6 mice (6–8 weeks) were maintained on a standard normal rodent diet (Synergy Bio, AIN-93M). All the mice used in this study were bred and raised in the animal facility of the First Affiliated Hospital of Sun Yat-sen University. High-salt diet and host sodium transporter inhibition experiments Mice (n = 5–6/ group) were fed a normal chow diet supplemented with 4% NaCl and drinking water contained 1% NaCl 9,10 for 28 days. This regimen provided an estimated daily NaCl intake of 0.22 g per 22 g mouse, based on assumed daily consumption of 4 g chow and 6 mL water (4 g chow × 0.04 + 6 mL water × 0.01 = 0.22 g NaCl/day). Control mice received the same normal chow diet and drank plain water. For Na + transporter inhibition experiment, mice (n = 5/group) maintained on the high-salt diet received cariporide (Aladdin Scientific, C286810) 54 or amiloride hydrochloride (APExBIO, B1884) 55 in 1% NaCl drinking water (1 mg/kg/day) for 5 consecutive days. At the end of experiments, mice were euthanized by cervical dislocation, and luminal contents were collected for Na⁺ and K⁺ quantification as described in the “Na + and K + quantification” section. Faecal microbiota transplantation (FMT) and bacteria monocolonisation Mice were randomly assigned as donors (n = 5/group) to a high-salt, high-protein (HSHP) or normal-salt, high-protein (NSHP) diet for 4 weeks. Fecal pellets collected after the intervention period were stored at − 80°C for metagenomic sequencing as described in the “Mouse fecal metagenomics sequencing” section. For FMT, recipient mice (n = 6) were pre-treated with a broad-spectrum antibiotic (Abx) cocktail 56 . Recipients on a high-protein background received daily oral gavage of faecal supernatant from HSHP or NSHP donors on days − 2 to + 1. Recipients on a normal-chow background underwent an identical protocol using donors fed a high-salt (HSD) or normal (NSD) chow diet on days − 2 to + 3. 2 days after FMT initiation, colitis was induced (day 0) in all recipients with 1.5–2% DSS water for 6–8 days. For monocolonisation, mice (n = 5/group) on normal chow received a 4-day Abx-cocktail, followed by daily oral gavage of E. coli CD09 or MG1655 (1.0 × 10⁹ CFU) for 5 days, concurrent with 8 days of 2.5% dextran sulfate sodium (DSS; MP Biomedicals, 0216011080) in drinking water. Disease Activity Index was monitored daily. After euthanasia, colons were flushed, and a 5-mm distal segment was fixed for histology. Statistical Analysis Statistical analyses were performed with Prism v.8.0 (GraphPad). For two-group comparisons, the statistical significance was determined by unpaired t test or nonparametric Mann-Whitney test as indicated. Multiple group comparisons were made by ANOVA for most of the studies. Each data point denotes individual human subject, animal, or biological replicate. Additional methods are provided in online supplemental information. Ethics declarations Declarations Ethics approval and consent to participate Study research protocols were reviewed and approved by the Ethical Committees of the First Affiliated Hospital of Sun Yat-sen University (2016[113], 2023[488]). Written informed consent was obtained from all participants. All animal studies were conducted under protocols approved by the Institutional Animal Care and Use Committee (IACUC) at the First Affiliated Hospital of Sun Yat-sen University (2024 [201]). Consent for publication Not applicable. Funding This work is supported by the National Natural Science Foundation of China (82341217 to M.C., 32570124 to Y.Z., 82270579 to R. F., 82370551to M.C.), National Key Research and Development Program (2023YFC2307004 to Y.Z.), Guangxi Natural Science Foundation (2024GXNSFFA010009 to R. F.). Availability of data and materials All study data are included in the article and/or SI Appendix. Data are available in a public, open access repository. Gene expression profiling data from high‑throughput sequencing was downloaded and reanalyzed from the following sources: GEO (GSE83687), EMBL‑EBI (E‑MTAB‑5464), and GSA‑Human (HRA007763). HMP IBD transcriptomic data were retrieved from https://ibdmdb.org/results. Metagenomic sequences for the PRISM, FAH‑SYSU, and Industrialized versus Hadza cohorts were downloaded and reanalyzed (NCBI BioProjects: PRJNA400072, PRJNA793776, PRJEB49206). The human fecal archaeal amplicon sequencing data, mouse fecal metagenomes, and E. coli CD09 genome generated in this study have been deposited in the European Nucleotide Archive (ENA) (Project: PRJEB110203). All bacterial strains, and reagents generated in this study are available from the lead contact upon completing Material Transfer Agreement. Competing interests The authors declare no competing interests. Authors' contributions Z. C., M.C., R. F., and Y. Z. designed research; W. Z., Y. G., Q. L., X. Z., W. Luo, W. Lai, performed research; R.M., X.W. and X.Z. collected clinical samples; W. Z., Q. L., X.Z., and Y. Z. analysed data; W. Z., Q. L., and Y. Z. wrote the initial paper; and Z. C., A.E. R. F., and X. Z. edited the manuscript. All authors read and approved the final manuscript. Acknowledgments We thank the First Affiliated Hospital of Sun Yat-sen University Research Computing for computational resources, maintenance, and support. 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Database Resources of the National Genomics Data Center, China National Center for Bioinformation in 2022. Nucleic acids research 50, D27-D38 (2022). Additional Declarations No competing interests reported. Supplementary Files SupplementaryTables.xlsx Supplinfo03252026.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 28 Apr, 2026 Reviewers invited by journal 15 Apr, 2026 Editor assigned by journal 27 Mar, 2026 Submission checks completed at journal 26 Mar, 2026 First submitted to journal 25 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8881149","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":625924037,"identity":"ea32c366-b851-4481-97eb-231dc372a926","order_by":0,"name":"Wenjie Zhang","email":"","orcid":"","institution":"First Affiliated Hospital of Sun Yat-sen University","correspondingAuthor":false,"prefix":"","firstName":"Wenjie","middleName":"","lastName":"Zhang","suffix":""},{"id":625924038,"identity":"8bcc2f56-2c84-451f-9b2a-41a244016c28","order_by":1,"name":"Yang Gao","email":"","orcid":"","institution":"First Affiliated Hospital of Sun Yat-sen University","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Gao","suffix":""},{"id":625924039,"identity":"5d596e3a-fd1a-4c3a-8b66-b0fcb69bfe9c","order_by":2,"name":"Qiaoyu Ling","email":"","orcid":"","institution":"First Affiliated Hospital of Sun Yat-sen University","correspondingAuthor":false,"prefix":"","firstName":"Qiaoyu","middleName":"","lastName":"Ling","suffix":""},{"id":625924040,"identity":"0e21598a-7260-4c03-8ec5-69b98a2c5e7d","order_by":3,"name":"Xiaochen Zhu","email":"","orcid":"","institution":"First Affiliated Hospital of Sun Yat-sen University","correspondingAuthor":false,"prefix":"","firstName":"Xiaochen","middleName":"","lastName":"Zhu","suffix":""},{"id":625924041,"identity":"9b9e06c1-9297-4fc9-995c-14de6b0aa84a","order_by":4,"name":"Wanrong Luo","email":"","orcid":"","institution":"Cleveland Clinic","correspondingAuthor":false,"prefix":"","firstName":"Wanrong","middleName":"","lastName":"Luo","suffix":""},{"id":625924048,"identity":"dfff94ea-3974-40e5-b4ac-f28682e10191","order_by":5,"name":"Ren Mao","email":"","orcid":"","institution":"First Affiliated Hospital of Sun Yat-sen University","correspondingAuthor":false,"prefix":"","firstName":"Ren","middleName":"","lastName":"Mao","suffix":""},{"id":625924054,"identity":"bd52635b-82d9-4cff-8aeb-61d1cf97a64b","order_by":6,"name":"Xueting Wu","email":"","orcid":"","institution":"First Affiliated Hospital of Sun Yat-sen University","correspondingAuthor":false,"prefix":"","firstName":"Xueting","middleName":"","lastName":"Wu","suffix":""},{"id":625924056,"identity":"adfd25ce-b95e-427a-95b2-2589c63b03fc","order_by":7,"name":"Wenhua Lai","email":"","orcid":"","institution":"First Affiliated Hospital of Sun Yat-sen University","correspondingAuthor":false,"prefix":"","firstName":"Wenhua","middleName":"","lastName":"Lai","suffix":""},{"id":625924059,"identity":"3dfb6b9e-ae08-42d8-9056-05d132654260","order_by":8,"name":"Ali H. 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The internal line represents the median. Significance was determined by non-parametric Mann–Whitney test.\u003c/p\u003e\n\u003cp\u003e(D) Diagnostic performance of fecal Na⁺and K⁺ levels for discriminating CD. CRP, C reactive protein; PLT, platelet count.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8881149/v1/3b157365aee0cd8fbc1088b8.png"},{"id":107619725,"identity":"4f83ee95-5b22-41a3-a73a-7f11afef93c6","added_by":"auto","created_at":"2026-04-23 09:31:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":966751,"visible":true,"origin":"","legend":"\u003cp\u003eLuminal Na⁺/K⁺ imbalance in CD is driven by dietary salt intake and impaired host Na\u003csup\u003e+\u003c/sup\u003e absorption.\u003c/p\u003e\n\u003cp\u003e(A, B) Schematic diagram showing the HSD challenge experimental design and sampling timeline (A). A HSD altered intestinal Na⁺ and K⁺ concentrations (B). Mean ± SD. Significance was determined by unpaired t test.\u003c/p\u003e\n\u003cp\u003e(C, D) Colonic Na\u003csup\u003e+\u003c/sup\u003e transporters (C). Analysis of mucosal transcriptomic data from multiple IBD cohorts demonstrates significant downregulation of key Na\u003csup\u003e+\u003c/sup\u003e transporter genes in CD patients compared to various control tissues (D). FAH-SYSU: non-disease; IBDMBD: symptomatic non-IBD; E-MTAB5464: non-disease; GSE83687: normal tissue from cancer patients. The internal line represents the median. Significance was determined by non-parametric Mann–Whitney test.\u003c/p\u003e\n\u003cp\u003e(E, F) Schematic diagram showing the experimental design and sampling timeline (E). Pharmacological inhibition of Na\u003csup\u003e+\u003c/sup\u003e transport in mice elevates luminal Na⁺ and reduces K⁺ (F). Mean ± SD. Significance was determined by unpaired t test.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8881149/v1/c9ab116ea9d5ee467d27ff4b.png"},{"id":107619726,"identity":"b5913907-2501-4df3-a8b8-11570fc71388","added_by":"auto","created_at":"2026-04-23 09:31:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":735586,"visible":true,"origin":"","legend":"\u003cp\u003eThe archaeome of CD patients is enriched in halophilic archaea.\u003c/p\u003e\n\u003cp\u003e(A) Archaeal community profiles in CD patients and healthy controls.\u003c/p\u003e\n\u003cp\u003e(B, C) CD individuals demonstrate elevated relative (B) and absolute (C) abundance of \u003cem\u003eHalorubrum\u003c/em\u003e and reduced \u003cem\u003eMethanobrevebacter\u003c/em\u003e. Significance was determined by non-parametric Mann–Whitney test.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8881149/v1/df7da4e68028c66015a16a99.png"},{"id":107619727,"identity":"1bd5e050-c336-429f-a7bf-cf152698f60e","added_by":"auto","created_at":"2026-04-23 09:31:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":645585,"visible":true,"origin":"","legend":"\u003cp\u003eThe gut microbiome in CD patients is enriched with bacterial salt-stress response genes.\u003c/p\u003e\n\u003cp\u003e(A) Schematic of microbial salt stress response pathways, including K\u003csup\u003e+\u003c/sup\u003e transporters and osmolyte transport/biosynthesis systems.\u003c/p\u003e\n\u003cp\u003e(B) Comparative analysis of microbial salt stress gene abundance in CD patients versus healthy controls across the FAH-SYSU and PRISM cohorts. Dot size represents the prevalence of a gene within a group, and color indicates the mean gene abundance (RPKM). G⁻, Gram-negative; G⁺, Gram-positive. \u003cem\u003ebcc ABC transporter\u003c/em\u003e,\u003cem\u003e betaine-choline-carnitine ABC transporter\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8881149/v1/ce8278f6e9f9a9d3eed314d6.png"},{"id":107707095,"identity":"83987d6d-718e-43d0-ac4d-3312f79194ca","added_by":"auto","created_at":"2026-04-24 09:19:30","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1422616,"visible":true,"origin":"","legend":"\u003cp\u003eSalt stress grants a selective advantage for salt-tolerant opportunistic pathogen.\u003c/p\u003e\n\u003cp\u003e(A) Phylogenetic analysis reveals that salt-stress response genes are widely distributed within the \u003cem\u003ePseudomonadota\u003c/em\u003e, \u003cem\u003eBacillota\u003c/em\u003e and \u003cem\u003eActinomycetota\u003c/em\u003ephyla among HMP reference genomes.\u003c/p\u003e\n\u003cp\u003e(B) Isolation and identification of salt tolerant bacterial strains from the colonic mucosa of CD individuals.\u003c/p\u003e\n\u003cp\u003e(C, D) Enhanced growth under high Na⁺ stress correlates with salt-tolerance genotypes. (C) Growth curves. (D) Presence of salt-stress response genes in the corresponding strains. Significance was determined by 2-way ANOVA.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8881149/v1/9af15cb82ee5f2ef45ea8dd7.png"},{"id":107705916,"identity":"49ce18aa-40bf-4ca1-a291-984be09d7d5a","added_by":"auto","created_at":"2026-04-24 09:15:43","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":815848,"visible":true,"origin":"","legend":"\u003cp\u003eThe high-salt, high-protein diet adapted gut microbiome exacerbated colitis.\u003c/p\u003e\n\u003cp\u003e(A) Experimental design, timeline, and sampling strategy for donor mice.\u003c/p\u003e\n\u003cp\u003e(B-D) Donor mice fed a high-salt, high-protein (HSHP) diet exhibit a thinner colonic mucus layer (B, C) and reduced \u003cem\u003emuc2\u003c/em\u003e gene expression (D) compared to mice on a normal-salt, high-protein (NSHP) diet. Mean ± SD. Significance was determined by unpaired t-test.\u003c/p\u003e\n\u003cp\u003e(E-G) The gut microbiota of HSHP-fed donors forms a distinct cluster (E), shows reduced Shannon diversity (F), and exhibits enriched microbial salt-stress gene abundance (G). Dot size represents gene prevalence and color indicates mean gene abundance (RPKM). Key genes are highlighted by function (K⁺ transport, osmolyte metabolism). G⁻, Gram-negative; G⁺, Gram-positive. \u003cem\u003ebcc ABC transporter\u003c/em\u003e,\u003cem\u003e betaine-choline-carnitine ABC transporter\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e(H-J) Experimental design for fecal microbiota transplantation (FMT), showing timeline and sampling at two time points (days 10 and 21) from HSHP-fed donors (H). Following FMT and DSS challenge, representative colon morphology (I), and colon length measurements (J) of recipient mice at endpoint. Mean ± SD. Significance was determined by unpaired t-test.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-8881149/v1/9c3013bf6bfe0a2f5c494082.png"},{"id":107705816,"identity":"67c60976-284f-42aa-8a2d-d286f68c2faa","added_by":"auto","created_at":"2026-04-24 09:15:23","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":787795,"visible":true,"origin":"","legend":"\u003cp\u003eIsolate \u003cem\u003eE. coli\u003c/em\u003e CD9 demonstrated higher salt tolerance and virulence \u003cem\u003ein vivo\u003c/em\u003e than lab strain compartment \u003cem\u003eE. coli\u003c/em\u003e MG1655.\u003c/p\u003e\n\u003cp\u003e(A, B) \u003cem\u003eE. coli \u003c/em\u003eCD9 exhibited enhanced growth under high Na⁺ stress as shown by growth curves (A) and doubling time (B). Mean ± SEM. Statistical significance was determined by one-way ANOVA with multiple comparisons for panel (B).\u003c/p\u003e\n\u003cp\u003e(C-I) Schematic diagram showing the experimental design, timeline and sampling strategy (C). Body weight (D) and disease activity index (E) were monitored following DSS administration. Colonic morphologies (F), colon length (G), representative H\u0026amp;E-stained colon sections (H) and histological assessment (I) at the termination of the experiment on day 8. Mean ± SD. Statistical significance was determined by unpaired t-test for panel G and I.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-8881149/v1/5cd1c58536522bb6e2204fe3.png"},{"id":107709227,"identity":"f31ddd23-8577-4ba1-b8ca-76d7bddc8663","added_by":"auto","created_at":"2026-04-24 09:35:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6230350,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8881149/v1/0391a4e1-0cdf-4a0f-a7d3-770ddc7b4230.pdf"},{"id":107619723,"identity":"53fce3a3-aa54-43ad-9337-2c422b5f2d13","added_by":"auto","created_at":"2026-04-23 09:31:58","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":369262,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTables.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8881149/v1/23653f3db8eeb585ba587d43.xlsx"},{"id":107706766,"identity":"1664cc3f-9c1c-4f5c-bd9e-241d69381727","added_by":"auto","created_at":"2026-04-24 09:18:41","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1400761,"visible":true,"origin":"","legend":"","description":"","filename":"Supplinfo03252026.docx","url":"https://assets-eu.researchsquare.com/files/rs-8881149/v1/2e7ed5003733be294133d7a3.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"A High-Salt Diet Promotes Colitis by Remodeling the Gut Microbiota Toward a Virulent, Osmotolerant State","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHigh-salt diets (HSDs) represent a significant risk factor for a spectrum of non-communicable chronic inflammatory diseases, including inflammatory bowel disease (IBD), hypertension, cardiovascular disease (CVD), and Alzheimer\u0026rsquo;s disease, and are associated with increased disease activity in multiple sclerosis\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. A wealth of epidemiological evidence strongly supports reducing salt consumption, aligning with the World Health Organization's recommendation to limit intake to \u0026lt;\u0026thinsp;5 g/day\u003csup\u003e4\u003c/sup\u003e. Nonetheless, global consumption frequently exceeds 10 g daily, more than 70% of the sodium we eat comes from packaged, prepared and restaurant foods due to salt added for flavoring, stabilizing, preserving and reducing bacterial risk\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. High dietary Na\u003csup\u003e+\u003c/sup\u003e elevates systemic and luminal Na\u003csup\u003e+\u003c/sup\u003e concentrations, impairing health through multiple distinct mechanisms. Initial research primarily focused on hemodynamic effects and direct pro-inflammatory immune activation, including vascular endothelial dysfunction, immune cell recruitment, and cytokine production\u003csup\u003e\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. More recent investigations have established the gut microbiota as a critical mediator of salt-sensitive pathology. The colon is a major absorptive and secretory organ for electrolytes. In healthy adults, the colon is to absorb 90% of 1.5\u0026ndash;2 L of electrolyte-rich small intestinal fluid that enters the colon daily\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. This flux thus represents a critical environmental stimulus for the gut microbiota. Recent studies demonstrated that a high-salt challenge depletes intestinal \u003cem\u003eLactobacillus\u003c/em\u003e spp., promotes the expansion of pro-inflammatory TH17 cells, lower beneficial metabolites \u003cem\u003ee.g.\u003c/em\u003e short chain fatty acids production\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, thereby positioning the gut microbiome as a viable therapeutic target for mitigating salt-associated disorders.\u003c/p\u003e \u003cp\u003eCrohn\u0026rsquo;s disease (CD), a subtype of IBD, is a chronic gastrointestinal inflammation imposes a substantial symptom burden and impairs quality of life \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Although the precise etiology of CD remains incompletely understood, disease onset is widely accepted to involve complex interactions between environmental factors, host genetics, gut microbiota, and immune responses\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. At the turn of the 21st century, IBD has become a global disease with accelerating incidence in newly industrialized countries in Africa, Asia, and South America, there is a pressing need for improved preventive and therapeutic strategies \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. While the Western diet and gut microbiome dysbiosis are primary drivers of the marked rise in CD incidence in recent decades, identifying modifiable dietary risk factors such as excessive salt intake is critical for developing improved preventive and therapeutic strategies. Prior studies found that \u003cem\u003eLactobacillus\u003c/em\u003e mediates HSD exacerbated colitis\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Owing to their documented health benefits, gastrointestinal \u003cem\u003eLactobacillus\u003c/em\u003e species are extensively studied and commonly used as probiotics. However, \u003cem\u003eLactobacillus\u003c/em\u003e species constitute a minor and highly variable fraction of the colonic microbiota, representing\u0026thinsp;~\u0026thinsp;0.01% of cultivable bacterial counts and are undetectable in nearly 25% of human fecal samples\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Furthermore, multi-cohort analyses in CD report inconsistent findings regarding \u003cem\u003eLactobacillus\u003c/em\u003e levels, with studies indicating both increases and decreases relative to healthy controls\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Consequently, the translational relevance of \u003cem\u003eLactobacillus\u003c/em\u003e-mediated, HSD-aggravated colitis to human CD pathophysiology remains unresolved.\u003c/p\u003e \u003cp\u003eWe reasoned that HSDs elevates luminal Na\u003csup\u003e+\u003c/sup\u003e concentration in the ileum and colon, thereby imposing significant salt and concomitant osmotic stress on the gut environment. Indeed, the gut archaeome in patients with colorectal cancer exhibits an enrichment of halophilic archaea compared to both adenoma patients and healthy individuals\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Osmolarity is a critical abiotic factor that profoundly shapes microbial ecosystems, and successful gut colonization depends on adaptation to such environmental pressures\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Prokaryotes employ two primary osmoregulatory strategies to thrive under salt-stressed conditions: the \"salt-in\" strategy, which maintains osmolarity through inorganic ion accumulation (\u003cem\u003ee.g.\u003c/em\u003e, K\u003csup\u003e+\u003c/sup\u003e), and the \"salt-out\" strategy, which utilizes compatible solutes to balance external pressure without disrupting intracellular function\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. While the salt tolerance of pathogens is well-characterized, the response of the broader human gut microbiome to salt-stress remains largely unknown.\u003c/p\u003e \u003cp\u003eIn this study, we analyzed a treatment na\u0026iuml;ve Crohn's disease cohort to profile fecal electrolytes (Na⁺, K⁺), quantify halophilic archaea, and assess metagenomic salt stress response genes. We characterized the growth phenotypes of representative species across a salinity gradient and investigated salt-stress adaptation at both polymicrobial community and single-strain levels, demonstrated that salt-tolerant opportunistic pathogens gain ecological advantage in the colon due to elevated luminal Na\u003csup\u003e+\u003c/sup\u003e levels. Collectively, our results provide novel mechanistic insights into how HSDs exacerbate colitis \u003cem\u003evia\u003c/em\u003e gut microbial community restructuring, revealing potential microbiome-targeted strategies for the prevention and treatment of HSDs imposed chronic inflammation diseases.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eFecal electrolyte analysis reveals a dysregulated luminal environment in CD\u003c/h2\u003e \u003cp\u003eThe colon dynamically regulates electrolyte homeostasis through absorptive and secretory processes\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. In CD, this balance is perturbed by the confluence of dietary habits, chronic diarrhea, mucosal inflammation, and impaired nutrient absorption\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. We therefore hypothesized that the fecal electrolyte profile, specifically the concentrations of Na\u003csup\u003e+\u003c/sup\u003e and K\u003csup\u003e+\u003c/sup\u003e, would be altered in CD. Analysis of stool samples from CD patients (n\u0026thinsp;=\u0026thinsp;108) and healthy individuals (n\u0026thinsp;=\u0026thinsp;124) confirmed this hypothesis. CD patients exhibited elevated Na\u003csup\u003e+\u003c/sup\u003e, reduced K\u003csup\u003e+\u003c/sup\u003e, and a consequently elevated Na\u003csup\u003e+\u003c/sup\u003e/K\u003csup\u003e+\u003c/sup\u003e ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-C). Fecal electrolyte profiles are significantly associated with CD phenotype and severity. Stratification of patients by disease behavior revealed that fecal K⁺ levels were approximately 1.5-fold higher in the non-stricturing, non-penetrating (B1) phenotype than in the stricturing (B2) or penetrating (B3) phenotypes (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). The fecal Na⁺/K⁺ ratio correlated positively with the Crohn's Disease Activity Index (CDAI), the gut inflammation marker fecal calprotectin, and systemic inflammation markers (neutrophil% and mean platelet volume), and correlated negatively with platelet count (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). In receiver operating characteristic (ROC) analysis, both fecal K\u003csup\u003e+\u003c/sup\u003e (AUC\u0026thinsp;=\u0026thinsp;0.87) and the Na\u003csup\u003e+\u003c/sup\u003e/K\u003csup\u003e+\u003c/sup\u003e ratio (AUC\u0026thinsp;=\u0026thinsp;0.79) demonstrated significantly greater discriminatory power for CD than platelet count (PLT, AUC\u0026thinsp;=\u0026thinsp;0.69) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e(A-C) Fecal analysis reveals significantly elevated Na⁺ (A), reduced K⁺ (B), and a consequently higher Na⁺/K⁺ ratio (C) in CD individuals compared to HCs. The internal line represents the median. Significance was determined by non-parametric Mann\u0026ndash;Whitney test.\u003c/p\u003e \u003cp\u003e(D) Diagnostic performance of fecal Na⁺ and K⁺ levels for discriminating CD. CRP, C reactive protein; PLT, platelet count.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDietary salt and impaired transport drive intestinal electrolyte dysregulation\u003c/h3\u003e\n\u003cp\u003eGiven the elevated fecal Na\u003csup\u003e+\u003c/sup\u003e and reduced K\u003csup\u003e+\u003c/sup\u003e levels observed in CD patients, we next investigated whether an HSD drives this luminal electrolyte imbalance. To test this, mice were fed an HSD for 4 weeks, after which luminal Na⁺ and K⁺ concentrations were quantified along the intestinal tract (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). While control mice maintained consistent Na⁺, K⁺, and Na⁺/K⁺ ratios from the small intestine to the colon, HSD feeding significantly perturbed this homeostasis (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA-C). Specifically, HSD led to elevated Na⁺ in the distal small intestine and cecum, a progressive decrease in K⁺ from the proximal small intestine to the colon, and a corresponding increase in the Na⁺/K⁺ ratio along the colonic axis compared to mice on NSD (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). However, the colonic content of HSD-fed mice did not fully recapitulate the human CD phenotype, which features high Na⁺ and low K⁺ (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-C). We reasoned that this discrepancy stems from the preserved electrolyte absorption capacity in healthy murine colon, a function known to be impaired in CD patients\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWe analyzed the expression of key Na\u003csup\u003e+\u003c/sup\u003e transporters, NHE1\u0026ndash;3, ENaC, and Na⁺/K⁺-ATPase, in intestinal biopsies from newly diagnosed CD patients (n\u0026thinsp;=\u0026thinsp;46) and non-disease controls (n\u0026thinsp;=\u0026thinsp;44) from the FAH-SYSU cohort\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Our analysis revealed a significant downregulation of NHE1 and NHE2 in inflamed CD mucosa, with a strong decreasing trend for Na⁺/K⁺-ATPase. This pattern of downregulation was corroborated in three independent IBD cohorts (GSE83687\u003csup\u003e24\u003c/sup\u003e, E-MTAB-5464\u003csup\u003e25\u003c/sup\u003e, HMP IBD\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D).\u003c/p\u003e \u003cp\u003eWe therefore hypothesized that the CD luminal Na\u003csup\u003e+\u003c/sup\u003e/K\u003csup\u003e+\u003c/sup\u003e profile results from the synergy of high dietary salt intake and impaired host electrolyte transport. To test this, we administered the Na\u003csup\u003e+\u003c/sup\u003e transport inhibitors amiloride and cariporide to mice\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Cariporide is a potent and selective NHE1 inhibitor, whereas amiloride is less specific, also inhibiting other NHE isoforms and ENaC. Consistent with our hypothesis, mice treated with amiloride exhibited an elevated fecal Na⁺/K⁺ ratio, driven by increased Na⁺ and decreased K⁺ concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, F; Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eD-G). This result directly validates that combined high dietary salt and impaired electrolyte transport synergistically generate a luminal environment of high Na\u003csup\u003e+\u003c/sup\u003e and low K\u003csup\u003e+\u003c/sup\u003e of CD.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e(A, B) Schematic diagram showing the HSD challenge experimental design and sampling timeline (A). A HSD altered intestinal Na⁺ and K⁺ concentrations (B). Mean \u0026plusmn; SD. Significance was determined by unpaired t test.\u003c/p\u003e \u003cp\u003e(C, D) Colonic Na\u003csup\u003e+\u003c/sup\u003e transporters (C). Analysis of mucosal transcriptomic data from multiple IBD cohorts demonstrates significant downregulation of key Na\u003csup\u003e+\u003c/sup\u003e transporter genes in CD patients compared to various control tissues (D). FAH-SYSU: non-disease; IBDMBD: symptomatic non-IBD; E-MTAB5464: non-disease; GSE83687: normal tissue from cancer patients. The internal line represents the median. Significance was determined by non-parametric Mann\u0026ndash;Whitney test.\u003c/p\u003e \u003cp\u003e(E, F) Schematic diagram showing the experimental design and sampling timeline (E). Pharmacological inhibition of Na\u003csup\u003e+\u003c/sup\u003e transport in mice elevates luminal Na⁺ and reduces K⁺ (F). Mean \u0026plusmn; SD. Significance was determined by unpaired t test.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCD fecal samples show increased abundance of halophilic archaeon\u003c/b\u003e \u003cb\u003eHalorubrum\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWhile the human archaeome remains largely uncharacterized, previous studies indicate the gut harbors a diverse archaeal community beyond the well-studied \u003cem\u003eMethanobrevibacter\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, with halophilic archaea reportedly enriched in colorectal cancer\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Indeed, \u003cem\u003eHalorubrum lipolyticum\u003c/em\u003e originally isolated from hypersaline environments like salt lakes, it is now recognized as a component of the diverse human gut archaeome\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. We therefore sought to characterize the archaeal composition in CD patients compared to healthy subjects. Using a conservative, archaea-specific 16S rRNA gene amplification and a bioinformatic pipeline, we profiled the fecal archaeome. Results revealed a diverse community dominated by the genera \u003cem\u003eMethanobrevibacter\u003c/em\u003e and a halophilic Archaea \u003cem\u003eHalorubrum\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Fecal samples from CD individuals (n\u0026thinsp;=\u0026thinsp;51) had significant enrichment of \u003cem\u003eHalorubrum\u003c/em\u003e and depletion of methanogenic archaea \u003cem\u003eMethanobrevibacter\u003c/em\u003e than HCs (n\u0026thinsp;=\u0026thinsp;131) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eTo validate these findings, we developed a multiplex droplet digital PCR (ddPCR) assay targeting both genera\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. The ddPCR results confirmed a reduction in the absolute abundance of \u003cem\u003eMethanobrevibacter\u003c/em\u003e and a concomitant increase in \u003cem\u003eHalorubrum\u003c/em\u003e in CD (n\u0026thinsp;=\u0026thinsp;50) than HCs (n\u0026thinsp;=\u0026thinsp;126), supporting the conclusion that a Na\u003csup\u003e+\u003c/sup\u003e-enriched colonic environment selectively enriches for halophilic archaea (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e(A) Archaeal community profiles in CD patients and healthy controls.\u003c/p\u003e \u003cp\u003e(B, C) CD individuals demonstrate elevated relative (B) and absolute (C) abundance of \u003cem\u003eHalorubrum\u003c/em\u003e and reduced \u003cem\u003eMethanobrevebacter\u003c/em\u003e. Significance was determined by non-parametric Mann\u0026ndash;Whitney test.\u003c/p\u003e\n\u003ch3\u003eA Na-enriched colon niche selects for a salt-tolerant microbiota\u003c/h3\u003e\n\u003cp\u003eThe initial phase of bacterial salt stress adaptation often involves the cytoplasmic accumulation of K⁺ ions, which serve as inorganic osmoprotectants to rapidly counterbalance external osmotic pressure\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. A subsequent, secondary response entails the biosynthesis and/or uptake of compatible solutes, such as carnitine, glycine betaine, trahelose, ectoine, proline \u003cem\u003eetc\u003c/em\u003e, which act as organic osmoprotectants to restore cell volume and turgor pressure\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. To systematically investigate this adaptive machinery, we compiled a comprehensive set of genes (clusters) (29) encoding: 1) transporters for active K⁺ uptake (5), 2) transporters for compatible solutes (20), and 3) enzymes for compatible solute biosynthesis (4) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, Supplementary Dataset 1).\u003c/p\u003e \u003cp\u003eWe conducted a comprehensive investigation into the salt-stress tolerance of the human gut microbiome, focusing on key genes responsible for active K\u003csup\u003e+\u003c/sup\u003e transport, compatible solute transport and biosynthesis. Our analysis was based on stool metagenomic samples from two independent IBD cohorts, FAH-SYSU and PRISM. We found that the abundances of gene (clusters) involved in salt adaptation, including K⁺ transporters (\u003cem\u003ekdpFABC\u003c/em\u003e, \u003cem\u003ekefBG\u003c/em\u003e, \u003cem\u003ekefCF\u003c/em\u003e), compatible solute transporters (\u003cem\u003eopuE\u003c/em\u003e/\u003cem\u003eputP\u003c/em\u003e, betaine-choline-carnitine ABC transporters, and \u003cem\u003ebcct\u003c/em\u003e family members), and the betaine biosynthesis cluster (\u003cem\u003ebetAB\u003c/em\u003e), were significantly enriched in the CD metagenome (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). This increasing trend was consistently observed in the independent PRISM cohort, though statistical significance was not reached for some gene clusters.\u003c/p\u003e \u003cp\u003eWe reasoned that the dramatic rise in dietary Na\u003csup\u003e+\u003c/sup\u003e intake following industrialization drives gut microbiota to a salt-tolerant structure. To test the hypothesis, we examined gut metagenomes from populations representing a spectrum of industrialization, including the Hadza hunter‑gatherers of Tanzania (n\u0026thinsp;=\u0026thinsp;125) and comparative populations from Nepal and California (n\u0026thinsp;=\u0026thinsp;54)\u003csup\u003e33\u003c/sup\u003e. Consistent with findings from FAH-SYSU and PRISM cohorts, several genes encoding K⁺ transporters (\u003cem\u003ekdpFABC\u003c/em\u003e) and compatible solute transporters (\u003cem\u003ebcc ABC transporter\u003c/em\u003e) reached statistical significance in the industralised populations (Nepal and California) comparing to non-industralised population (Hadza) (Figure S3), confirming the enrichment of salt‑stress response genes in industrialized populations.\u003c/p\u003e \n\u003ch3\u003ePhylogenetic distribution of salt tolerant human associated bacteria\u003c/h3\u003e\n\u003cp\u003eTo systematically evaluate the phylogenetic distribution of salinity-responsive genes in the human microbiome, we profiled their presence across 1,640 reference genomes from the Human Microbiome Project (HMP). This analysis revealed a significant enrichment of salt-stress response genes in opportunistic pathogens, particularly facultative and obligate aerobes from \u003cem\u003ePseudomonadota\u003c/em\u003e and \u003cem\u003eBacillota\u003c/em\u003e phyla, such as \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eProteus mirabilis\u003c/em\u003e, \u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e, and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, Supplementary Dataset 2). Skin is a high osmotic stress environment for bacteria due to low moisture and high salt concentrations from evaporated sweat. As expected, salt-stress tolerant bacteria were also observed in skin-associated members of the \u003cem\u003eActinomycetota\u003c/em\u003e phylum, including \u003cem\u003eCutibacterium acnes\u003c/em\u003e and \u003cem\u003eCorynebacterium\u003c/em\u003e species (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, Supplementary Dataset 2). Notably, 17.5% of genomes (287/1,640) harbored\u0026thinsp;\u0026ge;\u0026thinsp;4 salt-tolerance genes (Figure S4A). These genotypes were predominantly from the \u003cem\u003ePseudomonadota\u003c/em\u003e phylum and the \u003cem\u003eStaphylococcaceae\u003c/em\u003e and \u003cem\u003eEnterococcaceae\u003c/em\u003e families from the \u003cem\u003eBacillota\u003c/em\u003e phylum, taxa commonly enriched in the CD gut microbiome \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Conversely, bacterial families strongly associated with host health state, including \u003cem\u003eLachnospiraceae\u003c/em\u003e and \u003cem\u003eOscillospiraceae\u003c/em\u003e (\u003cem\u003eBacillota\u003c/em\u003e), \u003cem\u003ePrevotellaceae\u003c/em\u003e and \u003cem\u003eRikenellaceae\u003c/em\u003e (\u003cem\u003eBacteroidota\u003c/em\u003e), and \u003cem\u003eAkkermansiaceae\u003c/em\u003e (\u003cem\u003eVerrucomicrobiota\u003c/em\u003e) \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, typically harbored\u0026thinsp;\u0026lt;\u0026thinsp;4 salt-stress response genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e\n\u003ch3\u003eIsolation of salt-tolerant bacteria from CD mucosal biopsy\u003c/h3\u003e\n\u003cp\u003eGiven the observed enrichment of salt-stress response genes in the CD metagenome, we next sought to determine whether viable salt-tolerant bacteria colonise the CD intestinal mucosa. Mucosal biopsies from 6 CD patients were homogenized and plated on rich medium supplemented with 6% NaCl, a concentration that inhibits most bacteria but selectively permits the growth of halotolerant species, as established in standard salinity stress assays (\u003cem\u003ee.g.\u003c/em\u003e, 6.5% NaCl broth for \u003cem\u003eStaphylococusi\u003c/em\u003e). Consistent with our genomic predictions, we successfully isolated 9 halotolerant species, including 7 from the \u003cem\u003ePseudomonadota\u003c/em\u003e phylum and 2 from the \u003cem\u003eBacillota\u003c/em\u003e phylum (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSalinity stress provides an ecological advantage to salt tolerant bacteria\u003c/h2\u003e \u003cp\u003eBacteria respond to salt stress differently, with salt tolerant strains outcompete salt sensitive strains to gain ecological advantage in salt stressed environment. We hypothesized that strains harboring multiple salt-stress response genes would exhibit enhanced salt tolerance, manifesting as reduced growth inhibition under high Na\u003csup\u003e+\u003c/sup\u003e conditions. To test this, we cultured a panel of gut bacteria in media containing 0.5%, 1.5%, or 2.5% NaCl. The highest concentration was selected to approximate the mean fecal Na\u003csup\u003e+\u003c/sup\u003e concentration measured in individuals within the 4th quartile of CD patients. This concentration corresponds to 21.89 Na⁺ mg/g (dry stool) (Figure S5), equivalent to a 2.5% NaCl solution assuming 85% fecal water content.\u003c/p\u003e \u003cp\u003eConsistent with our hypothesis, elevated Na\u003csup\u003e+\u003c/sup\u003e concentration progressively inhibited the growth of various commensal gastrointestinal bacteria. The growth of commensal bacteria, \u003cem\u003eBacteroides ovatus\u003c/em\u003e, \u003cem\u003eClostridium butyricum, Bacteroides thetaiotaomicron\u003c/em\u003e, \u003cem\u003eAkkermansia muciniphila\u003c/em\u003e, \u003cem\u003eClostridium sporogenes\u003c/em\u003e, and probiotic \u003cem\u003eBifidobacterium longum\u003c/em\u003e were significantly inhibited by increasing NaCl in the medium, with \u003cem\u003eB. ovatus\u003c/em\u003e, and \u003cem\u003eA. muciniphila\u003c/em\u003e was completely inhibited at 1.5% and 2.5% NaCl. In contrast, \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eS. aureus\u003c/em\u003e, and \u003cem\u003eP. mirabilis\u003c/em\u003e, all of which carrying\u0026thinsp;\u0026gt;\u0026thinsp;=\u0026thinsp;4 salt-tolerance genes, maintained growth rates at 2.5% NaCl that were comparable to those observed at 0.5% (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D, Supplementary Dataset 3).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eThe salt-stress adapted microbiota exacerbates intestinal inflammation\u003c/h3\u003e\n\u003cp\u003eWe proposed that excess dietary salt leads to elevated gut luminal Na\u003csup\u003e+\u003c/sup\u003e levels, which promote salt tolerant microbial communities which worsen colon inflammation. To test this, mice received a high salt, high protein diet (HSHP), modeling a dietary pattern associated with CD \u003csup\u003e36\u003c/sup\u003e. After 21 days, these mice showed a thinner colon mucus layer and lower \u003cem\u003emuc2\u003c/em\u003e gene expression compared to mice on an isocaloric normal salt high protein diet (NSHP) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-D). Their gut microbiomes also formed distinct clusters (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE), exhibited reduced Shannon diversity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF), and showed an increasing trend in the abundance of salt-stress response genes. Among these, \u003cem\u003ekdpA\u003c/em\u003e, \u003cem\u003ekdpC\u003c/em\u003e, and \u003cem\u003ekimA\u003c/em\u003e reached statistical significance (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003eTo test whether a salt-stress adapted microbiota is sufficient to exacerbate inflammation, we performed fecal transplants. Donor mice were fed either a HSHP or NSHP. Fecal microbiota transplantation (FMT) experiments were performed using donors from each dietary group at 10 and 21 days\u0026rsquo; post-diet initiation. After inducing colitis, mice that received fecal transplant from HSHP donors consistently developed significantly shorter colons, indicating more severe inflammation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH-J). Notably, this effect required the combined high salt and high protein diet, as transplants from mice on a high salt normal chow diet post 10 and 28 days did not cause shorter colon length (Figure S6A-C). This demonstrates that a diet rich in both salt and protein shapes a pro-inflammatory gut microbiome capable of driving more severe colitis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e(A) Experimental design, timeline, and sampling strategy for donor mice.\u003c/p\u003e \u003cp\u003e(B-D) Donor mice fed a high-salt, high-protein (HSHP) diet exhibit a thinner colonic mucus layer (B, C) and reduced \u003cem\u003emuc2\u003c/em\u003e gene expression (D) compared to mice on a normal-salt, high-protein (NSHP) diet. Mean \u0026plusmn; SD. Significance was determined by unpaired t-test.\u003c/p\u003e \u003cp\u003e(E-G) The gut microbiota of HSHP-fed donors forms a distinct cluster (E), shows reduced Shannon diversity (F), and exhibits enriched microbial salt-stress gene abundance (G). Dot size represents gene prevalence and color indicates mean gene abundance (RPKM). Key genes are highlighted by function (K⁺ transport, osmolyte metabolism). G⁻, Gram-negative; G⁺, Gram-positive. \u003cem\u003ebcc ABC transporter\u003c/em\u003e, \u003cem\u003ebetaine-choline-carnitine ABC transporter\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e(H-J) Experimental design for fecal microbiota transplantation (FMT), showing timeline and sampling at two time points (days 10 and 21) from HSHP-fed donors (H). Following FMT and DSS challenge, representative colon morphology (I), and colon length measurements (J) of recipient mice at endpoint. Mean \u0026plusmn; SD. Significance was determined by unpaired t-test.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSalt tolerance in\u003c/b\u003e \u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003eassociates with pathogenicity in colitis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBacterial salt tolerance varied between strains. We compared the halotolerance of \u003cem\u003eE. coli\u003c/em\u003e CD09, a clinical isolate from this study, and the laboratory strain \u003cem\u003eE. coli\u003c/em\u003e MG1655 by culturing them in K\u003csup\u003e+\u003c/sup\u003e-depleted M9 medium with 0.5% or 2.5% NaCl. While both strains exhibited increased doubling times at 2.5% NaCl compared to 0.5%, \u003cem\u003eE. coli\u003c/em\u003e CD09 displayed significantly greater salt tolerance, indicated by significantly shorter doubling time at 2.5% NaCl (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, B). Despite sharing a core set of salt-stress response genes with the laboratory strain \u003cem\u003eE. coli\u003c/em\u003e MG1655 (Supplementary Dataset 3), the clinical isolate \u003cem\u003eE. coli\u003c/em\u003e CD09 exhibited significantly greater salt tolerance, indicating additional, uncharacterized genetic or regulatory mechanisms that likely contribute to its superior salt stress resistance.\u003c/p\u003e \u003cp\u003ePrevious studies demonstrated that bacteria salt tolerance is associated with infection, biofilm formation, antibiotic resistance and virulence \u003cem\u003eetc\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, hence we reasoned that enhanced salt tolerance of \u003cem\u003eE.coli\u003c/em\u003e CD09 was associated with increased pathogenicity \u003cem\u003ein vivo\u003c/em\u003e. Abx-treated mice were colonized with either \u003cem\u003eE. coli\u003c/em\u003e CD09 or MG1655 and then subjected to DSS-induced colitis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Mice colonized with the \u003cem\u003eE. coli\u003c/em\u003e CD09 isolate developed more severe disease, as indicated by significantly greater body weight loss, higher disease activity index scores, and exacerbated histopathological damage, including extensive mucosal erosion, crypt loss, and inflammatory cell infiltration (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD-I).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e(A, B) \u003cem\u003eE. coli\u003c/em\u003e CD9 exhibited enhanced growth under high Na⁺ stress as shown by growth curves (A) and doubling time (B). Mean \u0026plusmn; SEM. Statistical significance was determined by one-way ANOVA with multiple comparisons for panel (B).\u003c/p\u003e \u003cp\u003e(C-I) Schematic diagram showing the experimental design, timeline and sampling strategy (C). Body weight (D) and disease activity index (E) were monitored following DSS administration. Colonic morphologies (F), colon length (G), representative H\u0026amp;E-stained colon sections (H) and histological assessment (I) at the termination of the experiment on day 8. Mean \u0026plusmn; SD. Statistical significance was determined by unpaired t-test for panel G and I.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWhile elevated Na\u003csup\u003e+\u003c/sup\u003e levels directly impact immune responses, they also drive the gut microbial community toward a more virulent structure, thus having synergistic effects, leading to more severe disease. The composition and function of the gut microbiota are fundamentally shaped by the physicochemical environment, wherein each taxonomic unit proliferates only when local conditions meet its specific biochemical and metabolic requirements. While microbial salinity adaptation has been extensively investigated in environments of marine, estuary and salt lakes, the role of gut luminal salinity driven by Na\u003csup\u003e+\u003c/sup\u003e and K\u003csup\u003e+\u003c/sup\u003e concentrations has been largely overlooked. While previous work has centered on the salt-induced depletion of \u003cem\u003eLactobacillus\u003c/em\u003e in the gut, this study provides a more comprehensive view of microbiome restructuring.\u003c/p\u003e \u003cp\u003eTo better understand how to remediate diseases associated with both dysbiosis and environmental perturbation of the gut, it is crucial to establish the physical parameter ranges in healthy and perturbed environments, including the microenvironments along the intestine that are relevant to disease states. In CD, concurrent excessive salt intake and impaired Na\u003csup\u003e+\u003c/sup\u003e absorption generate a high Na\u003csup\u003e+\u003c/sup\u003e, low K\u003csup\u003e+\u003c/sup\u003e luminal environment that imposes significant salt stress on gut microbiota. Here, we characterized the salt-stress adaptation from three aspects: 1) halophilic archaeon \u003cem\u003eHalorubrum\u003c/em\u003e is enriched in CD patients indicated by both relative and absolute abundance quantification. \u003cem\u003eHalorubrum\u003c/em\u003e was detected by archaea specific and \u003cem\u003eHalorubrum\u003c/em\u003e specific primers, although it has not been cultured; 2) the enrichment of bacterial salt-stress response genes by metagenomic analysis; 3) isolation and culturing of halotolerant bacteria from CD biopsies. In summary, we demonstrate that the Na\u003csup\u003e+\u003c/sup\u003e-enriched luminal environment in CD drives a compositional shift toward a more salt-tolerant microbiota.\u003c/p\u003e \u003cp\u003eMicrobial salt tolerance is associated with pathogenicity at both the microbial community and individual strain levels. Colonization of mice with a salt-stress adapted microbiota or a more halotolerant \u003cem\u003eE. coli\u003c/em\u003e strain resulted in exacerbated colitis \u003cem\u003ein vivo\u003c/em\u003e. A variety of strategies were employed by varying microbial linage. Opportunistic pathogens from \u003cem\u003eEnterobacterales\u003c/em\u003e and \u003cem\u003eStaphylococcaceae\u003c/em\u003e are enriched in CD patients\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, which harbor multiple salt-stress response genes, hence are more sustained in high Na\u003csup\u003e+\u003c/sup\u003e medium, whereas commensal bacteria, typically enriched in HCs, such as strains from \u003cem\u003eClostridiaceae\u003c/em\u003e, \u003cem\u003eOscillospiraceae\u003c/em\u003e, and \u003cem\u003eLachnospiraceae\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e which carrying minimal salt-stress response genes, exhibit severe growth inhibition as Na\u003csup\u003e+\u003c/sup\u003e concentration rises. Salt stress is frequently linked to a broader environmental stress response, encompassing resistance to osmolarity, cold and acid shock. For instance, BCCT and ABC transporter families contribute significantly to cryotolerance\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Therefore, carrying salt-stress response genes enable them to survive in fluctuation environment and enhance infection\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Indeed, deletion of osmolyte transporters attenuated pathogenicity of \u003cem\u003eL. monocytogenes\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eShigella\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. In addition, bacteria senses salinity to trigger virulence mechanism\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Thus, inherent environmental stress tolerance, coupled with shared genetic regulation of virulence, potentiates the pathogenicity of salt-tolerant bacteria. Our findings reveal that the gut microbiome mediates the pro-inflammatory effects of a high salt diet through ecological mechanisms that extend beyond the depletion of \u003cem\u003eLactobacillus\u003c/em\u003e, identifying luminal salt stress induced microbial community restructuring as a key mechanism linking HSDs to colitis development. It is noteworthy that the pro-inflammatory effect of a salt adapted microbiota displayed dietary dependence. Transplantation from HSHP donors exacerbated colitis, while microbiota from high salt, normal chow donors did not, indicating that the dietary context determines salt-driven microbial pathogenicity.\u003c/p\u003e \u003cp\u003eGut microbes coevolve with their host. Excessive dietary Na\u003csup\u003e+\u003c/sup\u003e is a critical global health challenge, implicated in an estimated 1.89\u0026nbsp;million deaths annually and associated with a spectrum of chronic conditions, including cardiovascular disease and IBD\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Epidemiological evidence consistently identifies high salt consumption, characteristic of Western diets, as a significant risk factor for CD\u003csup\u003e3\u003c/sup\u003e. Pre-agricultural humans are estimated to have consumed only about 768 mg of Na\u003csup\u003e+\u003c/sup\u003e per day (~\u0026thinsp;2 g/day salt)\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Globally, people consume nearly double the recommended amount of Na\u003csup\u003e+\u003c/sup\u003e (5 g/day salt). The rise in Na\u003csup\u003e+\u003c/sup\u003e consumption since industrialization, largely driven by its pervasive use in processed and prepared foods, frequently results in intake that far exceeds physiological requirements. Historically, dietary salt was scarce. Consequently, non-industrialized populations such as the Hadza hunter-gatherers harbor a lower prevalence and abundance of bacterial genes that confer salt tolerance, including those for K\u003csup\u003e+\u003c/sup\u003e active transport and compatible solute transport, in comparison to industrialized populations. Their microbiomes are also enriched with taxa from the \u003cem\u003eSpirochaetota\u003c/em\u003e phylum that carry minimal salt-stress response gene\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. In contrast, opportunistic pathogens from the \u003cem\u003ePseudomonadota\u003c/em\u003e and \u003cem\u003eBacillota\u003c/em\u003e phyla, which typically carry\u0026thinsp;\u0026gt;\u0026thinsp;=\u0026thinsp;4 salt-stress response genes, are commonly enriched in conditions associated with excessive salt intake and Western diet, including IBD, CVD, and obesity\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Although we focused on CD in this study, this microbial restructuring may also contribute to other HSDs-linked diseases.\u003c/p\u003e \u003cp\u003eIn conclusion, we report that CD patients exhibit a gut microenvironment characterized by high Na\u003csup\u003e+\u003c/sup\u003e, low K\u003csup\u003e+\u003c/sup\u003e levels. This altered luminal salinity drives microbial adaptation toward salt tolerance, enhances virulence at both the community and strain levels, and ultimately exacerbates intestinal inflammation. Our findings not only establish a causal link between gut microbial stress adaptation and virulence but also highlight this adaptive pathway as a promising target for novel therapeutic interventions in CD management.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eHuman subjects\u003c/h2\u003e \u003cp\u003e All study protocols abided by the Declaration of Helsinki principles and were approved by Ethical Committees of the First Affiliated Hospital of Sun Yat-sen University. Intestinal biopsies and stool specimens were collected as part of the FAH-SYSU cohort study (2016[113], 2023[488]). Subject stool samples were collected at the FAH, SYSU gastroenterology clinic and stored at -80\u0026deg;C immediately. For culturing assays, tissue samples were collected and stored in cryogenic vials at -80\u0026deg;C until use \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eNa\u003csup\u003e+\u003c/sup\u003e and K\u003csup\u003e+\u003c/sup\u003e quantification\u003c/h2\u003e \u003cp\u003eIntestinal luminal contents and faecal samples were lyophilized for 12 hours. Approximately 8 mg of homogenized dry sample was weighed and digested in 1 mL of 3% nitric acid (HNO₃) overnight at room temperature with gentle agitation. Na⁺ and K⁺ concentrations in the digests were quantified \u003cem\u003evia\u003c/em\u003e flame photometry (ContrAA 800, Analytik Jena) or inductively coupled plasma optical emission spectrometry (Avio 500, PerkinElmer). Characteristic emission wavelengths of 589.9 nm for Na⁺ and 766.5 nm for K⁺ were used. All digestion and analytical steps were performed in plasticware to minimize exogenous ion contamination.\u003c/p\u003e \u003cp\u003e \u003cb\u003eArchaeome profiling and Droplet digital PCR (ddPCR) for\u003c/b\u003e \u003cb\u003eMethanobrevibacter\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eHalorubrum\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTotal microbial genomic DNA was extracted from faecal samples using the magnetic bead-based MagPure FFPE DNA/RNA Kit (Magen Biotechnology, D6364-02) and quantified spectrophotometrically (Implen GmbH, NanoPhotometer N60). Archaeal community profiling was performed following the protocol of Koskinen et al. (2017) \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Absolute quantification of \u003cem\u003eMethanobrevibacter\u003c/em\u003e and \u003cem\u003eHalorubrum\u003c/em\u003e was performed \u003cem\u003evia\u003c/em\u003e ddPCR using the \u003cem\u003emcrA\u003c/em\u003e and \u003cem\u003erpoB\u003c/em\u003e marker genes, respectively. Gene-specific primers and probes were designed using PrimerQuest and validated by BLAST analysis. Each 20 \u0026micro;L ddPCR reaction contained 10 \u0026micro;L of ddPCR Supermix (Bio-Rad, no dUTP, Bio-Rad, 1863024), 900 nM of each primer, 250 nM of probe, and ~\u0026thinsp;100 ng of faecal DNA. Non-template and positive controls (\u003cem\u003eM. smithii\u003c/em\u003e CCAM68 and \u003cem\u003eH. lacusprofundi\u003c/em\u003e ATCC 49239) were included.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eBacterial growth\u003c/h2\u003e \u003cp\u003eBacterial strains were revived from glycerol stocks on solid media at 37\u0026deg;C. After 24\u0026ndash;48 hours, single colonies were inoculated into liquid medium and grown overnight. Strains were cultured as follows: \u003cem\u003eBacteroides ovatus\u003c/em\u003e ATCC 8483 and \u003cem\u003eAkkermansia muciniphila\u003c/em\u003e BAA-835 in Brain Heart Infusion (BHI) medium (HKM Bio, 024053); \u003cem\u003eBifidobacterium longum\u003c/em\u003e JCM11341 in BBL medium (Hope Bio, HB8777); \u003cem\u003eBacteroides thetaiotaomicron\u003c/em\u003e ATCC 29148, \u003cem\u003eClostridium butyricum\u003c/em\u003e ATCC 19398, and \u003cem\u003eC. sporogenes\u003c/em\u003e ATCC 15579 in Chopped Meat medium (TOPBIO, MD155B) (all anaerobically). \u003cem\u003eEscherichia coli\u003c/em\u003e MG1655, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC 29213, and \u003cem\u003eProteus mirabilis\u003c/em\u003e GN2 were grown aerobically in Luria broth medium (Hope Bio, HB0128). For salt-tolerance assays, overnight cultures (1:100, \u003cem\u003ev/v\u003c/em\u003e) were grown in medium with 0.5%, 1.5%, or 2.5% NaCl (\u003cem\u003ew/v\u003c/em\u003e). Growth (OD₆₀₀) was measured at intervals in triplicate and analysed using a logistic growth model.\u003c/p\u003e \u003cp\u003e \u003cem\u003eE. coli\u003c/em\u003e strains MG1655 and CD09 were initially cultured in LB medium and then subcultured (1:100, \u003cem\u003ev/v\u003c/em\u003e) into a modified K\u003csup\u003e+\u003c/sup\u003e depleted M9 medium containing 4 mM Na₂HPO₄, 22 mM NaH₂PO₄, 18.5 mM NH₄Cl, 1 mM MgSO₄, 0.1 mM CaCl₂, 10 \u0026micro;g/mL thiamine, and 0.5% glucose. This defined medium was supplemented with either 0.5% or 2.5% NaCl (\u003cem\u003ew/v\u003c/em\u003e) to impose osmotic stress. Cultures were grown aerobically at 37\u0026deg;C with shaking (250 rpm), and bacterial growth was monitored by measuring the optical density at 600 nm (OD₆₀₀).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eBacteria isolation and identification\u003c/h2\u003e \u003cp\u003eApproximately 50 mg of colonic mucosal tissue from CD patients was homogenized in 500 \u0026micro;L PBS. The supernatant was serially diluted (10- and 100-fold) and plated on LB agar with 6% NaCl (\u003cem\u003ew/v\u003c/em\u003e). After 48 hours of aerobic incubation, distinct colonies were picked and smeared onto a MALDI target plate. Spots were treated with 1.0 \u0026micro;L of 70% formic acid, air-dried for 5 minutes, overlaid with matrix solution (α-cyano-4-hydroxycinnamic acid saturated in 50% acetonitrile with 5% trifluoroacetic acid, \u003cem\u003ew/v\u003c/em\u003e), and analyzed by MALDI-TOF/MS (Bruker Biotyper smart). Spectra were collected and compared against the manufacturer\u0026rsquo;s reference for identification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eAnalyses of salt tolerance associated genes in Human Microbiome project (HMP) references genomes\u003c/h2\u003e \u003cp\u003eHMP references genomes (1640 genomes as of October 30, 2025) were selected and analyzed through the IMG program on the Joint Genome Institute website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://img.jgi.doe.gov/\u003c/span\u003e\u003cspan address=\"https://img.jgi.doe.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Key genetic determinants for salt-stress adaptation, including K⁺ active transporters, osmoprotectant transporters, and biosynthetic enzymes, were systematically catalogued from the literature (Supplementary Dataset 1). Hits were manually inspected. Genomes carrying salt-stress response gene(s) were selected to generate a phylogenetic tree using phyloT (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://phylot.biobyte.de/\u003c/span\u003e\u003cspan address=\"https://phylot.biobyte.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) based on NCBI taxonomy and visualized using iTOL \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Genome and gene IMG ID are available in Supplementary Dataset 2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eMetagenomic data analysis\u003c/h2\u003e \u003cp\u003eWe used ShortBRED\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e to accurately profile the abundance of genes involved in the salt-stress response in metagenomes sourced from the FAH-SYSU (BioProject: PRJNA793776)\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e, PRISM (BioProject: PRJNA400072)\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e, Hadza Hunter-Gatherers (BioProject: PRJEB49206)\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e datasets. We initially compiled a set of identified bacterial salt-stress response genes as our query sequences (Supplementary Dataset 2). ShortBRED-Identify was employed to generate markers for these key bacterial salt-stress response gene sequences using UniRef90 (June, 2025) as a reference list with an 85% cluster ID threshold. These markers were applied in ShortBRED-Quantify to assess gene abundance in paired metagenomes, which had previously undergone quality control \u003cem\u003evia\u003c/em\u003e the KneadData workflow (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://huttenhower.sph.harvard.edu/kneaddata\u003c/span\u003e\u003cspan address=\"http://huttenhower.sph.harvard.edu/kneaddata\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The output from ShortBRED-Quantify was expressed as reads per kilobase per million mapped reads (RPKM).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eAnimal Studies\u003c/h2\u003e \u003cp\u003eMale SPF C57BL/6 mice (6\u0026ndash;8 weeks) were maintained on a standard normal rodent diet (Synergy Bio, AIN-93M). All the mice used in this study were bred and raised in the animal facility of the First Affiliated Hospital of Sun Yat-sen University.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eHigh-salt diet and host sodium transporter inhibition experiments\u003c/strong\u003e \u003cp\u003eMice (n\u0026thinsp;=\u0026thinsp;5\u0026ndash;6/ group) were fed a normal chow diet supplemented with 4% NaCl and drinking water contained 1% NaCl\u003csup\u003e9,10\u003c/sup\u003e for 28 days. This regimen provided an estimated daily NaCl intake of 0.22 g per 22 g mouse, based on assumed daily consumption of 4 g chow and 6 mL water (4 g chow \u0026times; 0.04\u0026thinsp;+\u0026thinsp;6 mL water \u0026times; 0.01\u0026thinsp;=\u0026thinsp;0.22 g NaCl/day). Control mice received the same normal chow diet and drank plain water. For Na\u003csup\u003e+\u003c/sup\u003e transporter inhibition experiment, mice (n\u0026thinsp;=\u0026thinsp;5/group) maintained on the high-salt diet received cariporide (Aladdin Scientific, C286810)\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e or amiloride hydrochloride (APExBIO, B1884)\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e in 1% NaCl drinking water (1 mg/kg/day) for 5 consecutive days. At the end of experiments, mice were euthanized by cervical dislocation, and luminal contents were collected for Na⁺ and K⁺ quantification as described in the \u0026ldquo;Na\u003csup\u003e+\u003c/sup\u003e and K\u003csup\u003e+\u003c/sup\u003e quantification\u0026rdquo; section.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eFaecal microbiota transplantation (FMT) and bacteria monocolonisation\u003c/strong\u003e \u003cp\u003eMice were randomly assigned as donors (n\u0026thinsp;=\u0026thinsp;5/group) to a high-salt, high-protein (HSHP) or normal-salt, high-protein (NSHP) diet for 4 weeks. Fecal pellets collected after the intervention period were stored at \u0026minus;\u0026thinsp;80\u0026deg;C for metagenomic sequencing as described in the \u0026ldquo;Mouse fecal metagenomics sequencing\u0026rdquo; section. For FMT, recipient mice (n\u0026thinsp;=\u0026thinsp;6) were pre-treated with a broad-spectrum antibiotic (Abx) cocktail\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. Recipients on a high-protein background received daily oral gavage of faecal supernatant from HSHP or NSHP donors on days\u0026thinsp;\u0026minus;\u0026thinsp;2 to +\u0026thinsp;1. Recipients on a normal-chow background underwent an identical protocol using donors fed a high-salt (HSD) or normal (NSD) chow diet on days\u0026thinsp;\u0026minus;\u0026thinsp;2 to +\u0026thinsp;3. 2 days after FMT initiation, colitis was induced (day 0) in all recipients with 1.5\u0026ndash;2% DSS water for 6\u0026ndash;8 days. For monocolonisation, mice (n\u0026thinsp;=\u0026thinsp;5/group) on normal chow received a 4-day Abx-cocktail, followed by daily oral gavage of \u003cem\u003eE. coli\u003c/em\u003e CD09 or MG1655 (1.0 \u0026times; 10⁹ CFU) for 5 days, concurrent with 8 days of 2.5% dextran sulfate sodium (DSS; MP Biomedicals, 0216011080) in drinking water. Disease Activity Index was monitored daily. After euthanasia, colons were flushed, and a 5-mm distal segment was fixed for histology.\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed with Prism v.8.0 (GraphPad). For two-group comparisons, the statistical significance was determined by unpaired t test or nonparametric Mann-Whitney test as indicated. Multiple group comparisons were made by ANOVA for most of the studies. Each data point denotes individual human subject, animal, or biological replicate.\u003c/p\u003e \u003cp\u003eAdditional methods are provided in online supplemental information.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eEthics declarations\u003c/h2\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStudy research protocols were reviewed and approved by the Ethical Committees of the First Affiliated Hospital of Sun Yat-sen University (2016[113], 2023[488]). Written informed consent was obtained from all participants. All animal studies were conducted under protocols approved by the Institutional Animal Care and Use Committee (IACUC) at the First Affiliated Hospital of Sun Yat-sen University (2024 [201]).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work is supported by the National Natural Science Foundation of China (82341217 to M.C., 32570124 to Y.Z., 82270579 to R. F., 82370551to M.C.), National Key Research and Development Program (2023YFC2307004 to Y.Z.), Guangxi Natural Science Foundation (2024GXNSFFA010009 to R. F.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll study data are included in the article and/or SI Appendix. Data are available in a public, open access repository. Gene expression profiling data from high‑throughput sequencing was downloaded and reanalyzed from the following sources: GEO (GSE83687), EMBL‑EBI (E‑MTAB‑5464), and GSA‑Human (HRA007763). HMP IBD transcriptomic data were retrieved from https://ibdmdb.org/results. Metagenomic sequences for the PRISM, FAH‑SYSU, and Industrialized \u003cem\u003eversus\u003c/em\u003e Hadza cohorts were downloaded and reanalyzed (NCBI BioProjects: PRJNA400072, PRJNA793776, PRJEB49206). The human fecal archaeal amplicon sequencing data, mouse fecal metagenomes, and \u003cem\u003eE. coli\u003c/em\u003e CD09 genome generated in this study have been deposited in the European Nucleotide Archive (ENA) (Project: PRJEB110203). All bacterial strains, and reagents generated in this study are available from the lead contact upon completing Material Transfer Agreement.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZ. C., M.C., R. F., and Y. Z. designed research; W. Z., Y. G., Q. L., X. Z., W. Luo, W. Lai, performed research; R.M., X.W. and X.Z. collected clinical samples; W. Z., Q. L., X.Z., and Y. Z. analysed data; W. Z., Q. L., and Y. Z. wrote the initial paper; and Z. C., A.E. R. F., and X. Z. edited the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the First Affiliated Hospital of Sun Yat-sen University Research Computing for computational resources, maintenance, and support. We are grateful to Dr. Yin Chen for his insightful discussion. We thank Dr. Huanlin Cui for guidance on archaeal primer design and \u003cem\u003eHalorubrum\u003c/em\u003e culture methodology.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBibbins-Domingo, K., \u003cem\u003eet al.\u003c/em\u003e Projected effect of dietary salt reductions on future cardiovascular disease. \u003cem\u003eThe New England journal of medicine\u003c/em\u003e 362, 590\u0026ndash;599 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAburto, N.J., \u003cem\u003eet al.\u003c/em\u003e Effect of lower sodium intake on health: systematic review and meta-analyses. \u003cem\u003eBmj\u003c/em\u003e 346, f1326 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKuang, R., O'Keefe, S.J.D., Ramos Del Aguila de Rivers, C., Koutroumpakis, F. \u0026amp; Binion, D.G. Is Salt at Fault? 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Database Resources of the National Genomics Data Center, China National Center for Bioinformation in 2022. \u003cem\u003eNucleic acids research\u003c/em\u003e 50, D27-D38 (2022).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"microbiome","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mbio","sideBox":"Learn more about [Microbiome](http://microbiomejournal.biomedcentral.com/)","snPcode":"40168","submissionUrl":"https://submission.nature.com/new-submission/40168/3","title":"Microbiome","twitterHandle":"@MicrobiomeJ","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Western diet, osmotic stress, coevolution, environmental adaptation, Halorubrum","lastPublishedDoi":"10.21203/rs.3.rs-8881149/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8881149/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eExcessive dietary salt disrupts the gut microbiota and is increasingly recognized as a contributor to the rising prevalence of chronic inflammatory diseases. In Crohn\u0026rsquo;s disease (CD), Western dietary patterns and gut microbiome dysbiosis are well‑established drivers of disease incidence. Identifying modifiable dietary factors, such as high salt intake, is therefore critical for developing improved preventive and therapeutic strategies. However, the precise effects of high-salt diets (HSDs) on the luminal electrolyte profile, the gut microbiome, and subsequent susceptibility to intestinal inflammation, however, remain poorly defined.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eUsing a treatment‑na\u0026iuml;ve cohort, we show that CD patients exhibit a distinct fecal electrolyte profile characterized by elevated Na⁺ and reduced K⁺, which correlates with markers of both gut and systemic inflammation. Relative and absolute quantification further reveal increased abundance of the halophilic archaeon \u003cem\u003eHalorubrum\u003c/em\u003e and a corresponding decrease in \u003cem\u003eMethanobrevibacter\u003c/em\u003e. Systematic profiling of salt‑stress associated functional genes, including those involved in K⁺ transport, compatible solute transport and biosynthesis, revealed that the CD gut microbiome is enriched with salt stress response genes. A similar enrichment trend was observed in industrialized populations compared to the Hadza hunter‑gatherer communities. Phylogenetic and growth assays demonstrated that these genetic determinants are more prevalent in opportunistic pathogens, which consequently exhibit greater resistance to growth inhibition under high Na⁺ conditions. Indeed, using high‑salt selective media, we isolated salt‑tolerant opportunistic pathogens from colonic biopsies of CD patients. Through \u003cem\u003ein vivo\u003c/em\u003e fecal microbiota transplantation, we demonstrate that a high‑Na⁺ adapted microbial community exacerbates colitis. Salt tolerance and its associated pathogenicity vary across strains. The \u003cem\u003eE. coli\u003c/em\u003e CD09 isolate from CD patients exhibited greater salt tolerance than the model strain \u003cem\u003eE. coli\u003c/em\u003e MG1655, and mice inoculated with CD09 developed more severe colitis.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eCollectively, our findings elucidate a mechanism by which HSDs exacerbate intestinal colitis through restructuring of the gut microbial community. A high‑Na⁺ luminal environment drives microbial adaptation toward salt tolerance, amplifies virulence at both the community and single‑strain levels, and ultimately exacerbates colitis. Thus, the gut microbiota represents a promising therapeutic target for preventing and mitigating salt‑associated chronic inflammatory diseases.\u003c/p\u003e","manuscriptTitle":"A High-Salt Diet Promotes Colitis by Remodeling the Gut Microbiota Toward a Virulent, Osmotolerant State","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-23 09:31:53","doi":"10.21203/rs.3.rs-8881149/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"279871148411050764980516187806363279039","date":"2026-04-28T16:53:54+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-15T15:55:30+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-27T19:52:35+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-26T21:53:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microbiome","date":"2026-03-25T15:22:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"microbiome","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mbio","sideBox":"Learn more about [Microbiome](http://microbiomejournal.biomedcentral.com/)","snPcode":"40168","submissionUrl":"https://submission.nature.com/new-submission/40168/3","title":"Microbiome","twitterHandle":"@MicrobiomeJ","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"64887d0d-ce24-4bdc-9dd3-9f562c412c83","owner":[],"postedDate":"April 23rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-23T09:31:53+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-23 09:31:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8881149","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8881149","identity":"rs-8881149","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}