Bowel preparation-induced changes in the gut environment facilitate pathogen and pathobiont colonization

doi:10.21203/rs.3.rs-4940942/v1

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Bowel preparation-induced changes in the gut environment facilitate pathogen and pathobiont colonization

https://doi.org/10.21203/rs.3.rs-4940942/v1

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Bowel preparation is a common procedure performed millions of times each year to clear the intestinal tract ahead of colonoscopy. During this procedure, a laxative is administered to clear the intestinal contents, which also results in the removal of the mucus layer that normally protects the underlying epithelium. While bowel preparation is generally considered safe in healthy individuals, with the microbiota typically recovering fully, it remains unclear whether pathogenic bacteria can exploit the environmental changes induced by this procedure to cause disease. In this study, we investigated the impact of bowel preparation on the susceptibility of mice to oral infection by the bacterial pathogen Salmonella enterica serovar Typhimurium. We found that post-bowel preparation, in the absence of antibiotic treatment, Salmonella robustly colonized, expanded in the gut and translocated to extraintestinal organs, while vehicle-treated mice were protected from infection. Colonization was effective at doses as low as 1,000 CFU and was independent of Salmonella motility, indicating that bowel preparation disrupts intestinal colonization resistance against this common enteric pathogen. We also discovered that pathobionts common in patients with inflammatory bowel disease (IBD) are highly resistant to osmotic perturbation, suggesting they may take advantage of bowel preparation to expand. In a human IBD microbiota mouse model, resident pathobionts translocated to extraintestinal organs post-bowel preparation, whereas this was not seen in mice with a conventional or healthy human microbiota. These results reveal the ability of pathogens and pathobionts to exploit disruptions in the gut environment and further elucidate the importance of the mucus layer and commensal microbiota in preventing enteric pathogen infection.

Biological sciences/Microbiology/Microbial communities/Microbiome

Biological sciences/Microbiology/Pathogens

Colonoscopies are crucial non-surgical procedures used by gastroenterologists to diagnose and monitor diseases in the distal gastrointestinal (GI) tract. To ensure the accuracy and safety of these examinations, effective bowel preparation (bowel prep) is essential. Bowel prep involves cleansing the intestinal tract of luminal contents ahead of colonoscopy, typically using derivatives of polyethylene glycol (PEG), an osmotic laxative commonly sold over the counter and administered in large volumes before the procedure^1,2. In the United States alone, an estimated 14 million colonoscopies are performed yearly³, highlighting the importance and prevalence of this procedure. Bowel prep has been shown to cause significant short-term disruptions to the gut microbiota, but in healthy individuals, the microbiota composition usually returns to normal within days^4,5. However, the impact of bowel prep on pathogen colonization in the gut is not clear. Specifically, the potential for disease-causing bacteria to exploit the disrupted gut environment following bowel prep remains unknown.

This concern is particularly relevant for patients with inflammatory bowel disease (IBD), comprising both Crohn’s disease (CD), and ulcerative colitis (UC). These individuals often harbor microbial species that can act as pathogens under certain conditions. These bacteria, known as pathobionts, are thought to worsen inflammation in IBD^6,7. Moreover, patients with IBD undergo more frequent colonoscopies than the general population to monitor disease progression, as intestinal mucosal changes are predictive of clinical recurrence⁸. Previous studies have shown that bowel prep may differentially affect the microbiota in patients with IBD as compared to healthy individuals, leading to longer-term changes in microbiota composition^9,10 as well as adverse effects that have not been mechanistically linked to the procedure itself^11–16.

Despite the widespread use of bowel prep, its effects on the gut environment, and its potential to facilitate pathogen colonization remain poorly understood. In previous work, we showed that laxative treatment over multiple days alters the gut microbiota and selects for species capable of thriving in high osmolality environments^17,18. These findings are corroborated by other studies which show that laxative treatment leads to increased susceptibility to pathogens^19–21. This study builds on these findings by investigating the effects of acute laxative treatment via bowel prep with a time and spatial resolution not achievable in human studies. Specifically, we examine the impacts of bowel prep on the gut microbiota, the local intestinal environment, and on the host’s resistance against pathogen colonization during the initial hours following bowel prep.

We chose Salmonella enterica, an important and well-characterized bacterial pathogen that can infect mice, but in a manner dependent on the microbiota as well as the intestinal environment^22–24. Specifically, S. enterica serovar Typhimurium (Salmonella) requires disruption of the microbiota, such as via antibiotic treatment, to robustly colonize the intestines of mice and cause gastroenteritis^25,26. Our results show that bowel prep can eliminate the natural microbiota's protection against Salmonella invasion, without the need for antibiotic treatment or other perturbations. Remarkably, Salmonella can infect bowel prep-treated mice at very low infectious doses and up to two days after the procedure. Additionally, we found that colonization was independent of Salmonella motility, suggesting that the inadvertent stripping of the GI mucus layer during bowel prep reduces the normal barriers to infection. Finally, in gnotobiotic mice colonized with human IBD microbiotas and treated with bowel prep, we observed persistent systemic translocation of bacteria recognized as pathobionts. This was not recapitulated in mice with a healthy human or mouse conventional microbiota.

These findings provide a novel model of Salmonella colonization facilitated by bowel prep and highlight the potential for severe bouts of malabsorption to promote pathobiont colonization in pathobiont-rich microbiotas. This study underscores the impact of bowel preparation on reducing the microbiota's natural colonization resistance against pathogens through multiple mechanisms: increased abundance of osmotically resistant pathobionts, depletion of the mucus layer, and reduction of competition by the commensal microbiota.

Bowel prep disrupts the gut environment.

To investigate the effects of bowel prep on the gut environment, we characterized a murine bowel prep method previously used to facilitate the transplantation of human microbes into mice²⁷. Mice with a conventional microbiota were fasted and then orally gavaged 4 times with PEG dissolved in water (bowel prep), while controls received water as the vehicle alone (Fig. 1A). Mice were euthanized six hours post-bowel prep, and intestinal tissues and contents were collected. Bowel prep-treated mice had an average cecal osmolality that was 1.8-fold higher than vehicle-treated controls (725 vs. 437 mOsm/Kg, p < 0.001) (Fig. 1B) and a higher cecal mass, indicative of osmotic diarrhea and increased luminal contents during bowel prep (Fig. S1A).

Previously, we showed that long-term, low-concentration exposure to PEG laxatives compromised the integrity of the intestinal mucus layer¹⁷, an essential structure for preventing invasion of bacteria into the mucosa. To investigate the impact of a shorter-term but higher- concentration PEG exposure on the mucosal lining, we fixed, stained and imaged the cecum and colon mucosal layers of bowel prep- and vehicle- treated mice. In vehicle-treated mice, the distal colon displayed the expected thick, continuous mucus layer that provides a barrier between the gut microbiota and the epithelium^17,28,29; however, this layer was largely depleted in the bowel prep-treated mice (Fig. 1C). We quantified the percentage of the epithelium covered with mucus, and the average thickness of the mucus layer. We found that mucus coverage and thickness decreased by approximately 64% and 74% in the distal colon of bowel prep-treated mice, respectively (79.1% covered vs 28.7%, p = 0.02; 22.8 µm thickness vs 6.0 µm, p = 0.02) (Fig. 1C, S1B). Upstream in the cecum, we observed that the mucus layer in healthy mice was more hydrated and patchier, as expected^29,30, while it was also compromised in bowel prep-treated mice, with an approximate 85% decrease in mucus coverage and 81% reduction in mucus layer thickness (48.8% covered vs 9.2%, p = 0.02; 8.3 µm thickness vs 1.2 µm, p = 0.03) (Fig. 1D, S1B). These findings indicate that even short-term, high-concentration PEG exposure can severely disrupt the protective mucus barrier in the gut and potentially impair the gut's natural defenses against pathogens.

Hematoxylin and eosin (H&E) staining also showed that while no immune cell infiltration was observed post-bowel prep, the cecal mucosa thickness was significantly reduced (Fig. 1E, S1C). Furthermore, this reduction was not observed in the distal colon (Fig. 1E), suggesting that water excretion in response to bowel prep is not uniform throughout the gut. We next measured how the microbiota was affected in these mice. Culturable anaerobic bacterial loads were reduced in the distal colon and small intestine but did not change in the cecum (Fig. 1F, Fig. S1D). In contrast, aerobic bacterial loads were largely unchanged in the distal colon and small intestine but increased approximately 17-fold in the cecum (Fig. 1F, Fig. S1D). Compositionally, we found that the cecal microbiota, as measured by 16S rRNA sequencing, was largely unchanged in mice 6 hours post-bowel prep when compared to vehicle-treated controls (Fig. 1G). Combined with the changes in bacterial loads, these data suggest that 16S rRNA sequencing is measuring a portion of the microbiota that is either not culturable or that is no longer alive post-bowel prep. Furthermore, when we measured individual short-chain fatty acid (SCFA) levels in cecal contents, we found their concentrations were significantly reduced in bowel prep-treated mice as compared to vehicle-treated controls (Fig. 1H, S1E). This reduction in SCFA levels, despite not impacting cecal pH (Fig. S1F), suggests an impact of bowel prep on microbial metabolism and gut homeostasis.

Bowel prep promotes Salmonella colonization

Having observed changes in the mucus layer and SCFA levels, we hypothesized that osmotically resistant enteric pathogens would be able to bypass colonization resistance more easily in a post-bowel prep gut environment than in normal conditions. Salmonella is an enteric bacterial pathogen known to withstand osmotic stress in high-salt environments³¹. PEG used in bowel prep is a hydrophilic polyether that exerts more detrimental effects on bacterial growth than salts (specifically sodium chloride) at the same osmolality¹⁷. We therefore measured Salmonella growth rates in vitro in media adjusted with PEG to various osmolality levels. At an osmolality comparable to that observed in the post-bowel prep gut (~ 800 mOsm/Kg), Salmonella growth was indistinguishable from growth under normal conditions (~ 450 mOsm/Kg, Fig. 2A). Having measured Salmonella’s tolerance to osmolality levels in vitro, we next challenged bowel prep- and vehicle- treated mice with 10⁶ CFU Salmonella (Fig. 2B). While vehicle-treated mice showed no detectable levels of Salmonella in their feces post-treatment, bowel prep-treated mice showed high levels of colonization (Fig. 2C). Specifically, fecal Salmonella levels reached 2 x 10⁹ CFU/mL by one day post-inoculation; these levels were maintained for at least three days post-inoculation, indicating active expansion in the intestinal tract (Fig. 2C) that was not sex dependent (Fig. S2A). These results suggest that disruptions to the gut environment post-bowel prep facilitate Salmonella colonization and growth.

Given Salmonella’s strong ability to colonize the post-bowel prep mouse gut, we measured whether it could translocate to extraintestinal organs. We identified significant colonization in the mesenteric lymph nodes, spleen and liver by three days post-inoculation (4.2 x 10⁶, 4.2 x 10⁴, 3.2 x 10⁴ CFU/g, respectively) (Fig. 2D). We next used H&E staining to visualize the host inflammatory response and to assess tissue integrity in the cecum and distal colon: the cecum was strongly affected by Salmonella infection, with pathological scores in this site significantly increased compared to vehicle-treated mice (Fig. 2E,F).

The standard model for Salmonella infection involves pre-treating mice with antibiotics to deplete colonization resistance by the native microbiota³². We therefore compared the Salmonella load in bowel prep-treated mice to the burdens found using the standard streptomycin infection model³² (Fig. S2B-F). While the maximum fecal burdens were 10-fold lower than in mice treated with streptomycin (Fig. S2B), translocated Salmonella levels were comparable in the extraintestinal organs (Fig. 2D, S2D). This indicates that bowel prep alone, without antibiotics, can dramatically facilitate Salmonella expansion in the gut and its translocation to extraintestinal sites.

We next investigated the minimum dose required for Salmonella to colonize the post-bowel prep gut. We found that inoculation with as few as 1,000 CFU led to most mice becoming colonized while vehicle-treated mice were resistant to colonization at doses three orders of magnitude higher (Fig. 2G, 2C). Moreover, Salmonella translocation levels largely increased with administered dose (Fig. 2H). These findings suggest a dose-dependent relationship between Salmonella inoculation dose and ultimate colonization levels post-bowel prep.

Timing of Salmonella infection post-bowel prep highlights the importance of the microbiota in colonization resistance

To investigate the medium-term effects of bowel prep on the gut environment and the subsequent disruption to colonization resistance, we challenged the treated mice with Salmonella at various times post-treatment and collected tissues 3 days post inoculation. As the cecum was strongly affected by Salmonella colonization post-bowel prep (Fig. 2E,F), we investigated how the cecal environment, including osmolality, microbiota composition, SCFA concentrations, and mucus layer thickness, changed at different time points post-bowel prep. Cecal osmolality recovered rapidly post-bowel prep and was indistinguishable from untreated mice by 24 hours post-bowel prep (Fig. 3A). Furthermore, cecal mucus coverage and thickness were also recovered to baseline by 24 hours post bowel prep (Fig. 3B, S3A-C). However, the cecal microbiota composition was still altered at least 48 hours post-bowel prep (Fig. 3C), with lower species diversity at the 24- and 48- hour time points compared to untreated mice (Fig. 3D). Finally, we found that SCFA concentrations were depleted in bowel prep-treated mice for at least 24 hours after treatment (Fig. 3E, S3D), and particularly, the levels of butyrate were still significantly lower at 24 hours post-bowel prep.

To investigate whether these changes to the gut environment and microbiota impact colonization resistance, we challenged bowel prep-treated mice with Salmonella at 6, 24, and 48 hours post-bowel prep (Fig. 3F). We found that Salmonella levels in the feces and mesenteric lymph nodes were similarly high at the 6- and 24- hour timepoints, but were significantly reduced by the 48-hour timepoint (Fig. 3G,H). The strong colonization of Salmonella observed while the SCFA levels are depleted in the cecum suggests that microbiota parameters play an important role in colonization resistance against Salmonella.

Flagellar motility is not required for Salmonella expansion post-bowel prep

Our data indicates that the strongest Salmonella colonization occurs at the height of bowel prep, around 6 hours post-procedure, at a time when the mucus layer is most impaired (Fig. 3). It has previously been shown that mice with a depleted mucus layer suffer impaired colonization resistance against Salmonella³³. Furthermore, flagellar propulsion has been shown to be essential for Salmonella, and other bacterial pathogens to penetrate the mucus layer and infect the underlying epithelium^34,35. We therefore reasoned that a non-motile mutant of Salmonella might be able to colonize robustly post-bowel prep. To test this hypothesis, we challenged bowel prep- or vehicle- treated mice with wildtype Salmonella or a non-motile ΔflhD mutant, which is missing the gene responsible for control of flagellar production³⁶ but is similarly resilient against osmotic perturbation (Fig. S4). We found that fecal levels of Salmonella ΔflhD post-bowel prep were comparable to those of wild type Salmonella by one day post-inoculation (Fig. 4A,B). Interestingly, Salmonella ΔflhD was able to translocate to the mesenteric lymph nodes but had limited translocation to the spleen and liver when compared to wildtype (Fig. 4C, 2D). These observations suggest that the disruption of the mucus layer following bowel prep enabled invasive interactions between Salmonella and the host epithelium without the requirement for motility.

Bowel prep rapidly alters the gut environment without immediate changes in host gene expression

To investigate whether a decrease in host mucus production contributes to the depletion of the mucus layer and allows for infection, we isolated RNA from mouse cecal tissue from untreated controls (baseline, collected at the 6-hour timepoint), 6, and 72 hours post-bowel prep, and performed transcriptomics and differential expression analysis (Fig. 5, S5). At 6 hours post-bowel prep, we found that gene expression was unchanged compared to baseline (Fig. 5A). This indicates that the differences observed in the gut environment at this early time point (Fig. 1) were driven by the impact of the bowel prep treatment rather than by a host response, and highlights how rapidly bowel prep (as opposed to host response) affects the gut environment during times of heightened vulnerability to pathogen infection. Importantly, the transcription of genes involved in mucus layer production, antimicrobial peptide production, tight junction maintenance and cellular stress responses were largely unchanged both at the height of osmotic diarrhea (6 hours post-bowel prep) as well as 72 hours post-bowel prep (Fig. 5B). Notable exceptions were hk2 (involved in metabolic response to stress³⁷), slc5a8 (an SCFA transporter³⁸), and cldn4 (a component of tight junction strands involved in paracellular permeability³⁹) which increased in expression at 72 hours as compared to 6 hours post-bowel prep (Fig. 5B). Furthermore, nlrp6 (a member of the NOD-like receptor family and cytosolic innate immune sensor that recognizes microbe‐associated molecular patterns⁴⁰) and slc26a3 (involved in response to hyperosmotic stress⁴¹) were downregulated at 72 hours as compared to 6 hours post-bowel prep (Fig. 5B). As expected, at 72 hours post-bowel prep there were significant changes in several genes involved in regulation of the circadian rhythm compared to baseline (Fig. S5B).

IBD pathobionts are resilient to osmotic perturbation, translocate and persist in mesenteric lymph nodes post-bowel prep

Given that bowel prep enhanced the ability of exogenously introduced Salmonella to escape the gut and reach extraintestinal organs, we investigated whether bacteria already present in the microbiota could also exploit the disrupted environment to translocate to extraintestinal organs. Specifically, pathobionts are known to exploit a disrupted gut habitat and are often facultative anaerobes^47–49; osmotically tolerant strains could therefore potentially flourish in the post-bowel prep gut environment. We tested 130 potential pathobionts belonging to 7 bacterial genera (Proteus, Morganella, Kluvvera, Klebsiella, Escherichia, Enterobacter and Citrobacter^47–49) which were isolated from patients with ulcerative colitis. We grew these strains in vitro in multiple osmolalities but with otherwise consistent nutritional availability, using a previously described high throughput growth curve assay¹⁸. Unlike commensal bacteria, which display limited resilience to increased osmolality¹⁸, the pathobionts tested showed strong growth at osmotic levels experienced in the gut post-bowel prep (< 890 mOsm/Kg) in both anaerobic and aerobic conditions, with most strains able to grow above 1100 mOsm/Kg (Fig. 6A, Fig. S6A). These results indicate that pathobionts can thrive under the osmotic conditions created by bowel prep and suggest that they may be able to outcompete osmotically sensitive commensals.

We next asked whether pathobionts' relatively high osmotic resistance could lead to a shift in community composition in conditions of laxative-induced osmotic stress. We grew a human UC microbiota in vitro in basal and PEG laxative osmotic stress conditions (400 and 800 mOsm/Kg, respectively) and analyzed community composition via 16S rRNA sequencing. Indeed, we observed a shift in community composition under high osmolality conditions (Fig. 6B), including an increased abundance in the family Enterobacteriaceae. Specifically, the genera Morganella and Escherichia-Shigella were increased in abundance in vitro (Fig. 6C), indicating that even in a complex community these pathobionts had a competitive advantage in high osmolality over the rest of the microbiota.

To further investigate whether pathobionts can benefit from the post-bowel prep gut environment, we inoculated germ-free mice with the same UC microbiota tested in vitro, bred the mice, and performed bowel prep on the offspring (Fig. S6B). We then measured translocation to extraintestinal organs and found that while translocation was most severe 24 hours post-bowel prep, bacterial presence was sustained in the lymph nodes even two weeks post-procedure (Fig. 6D). In contrast, bowel prep in gnotobiotic mice colonized with a healthy human microbiota, as well as mice with a conventional mouse microbiota, did not lead to translocation to extraintestinal organs post-bowel prep (Fig. 6D). We sequenced the bacteria that had translocated to the mesenteric lymph nodes in the UC microbiota-colonized mice and identified genera recognized as pathobionts in these organs that persisted even two weeks post-bowel prep (Fig. 6E). We performed 16S rRNA sequencing on the fecal microbiota and found that most translocated taxa increased in relative abundance 24 hours post bowel prep, suggesting they had a competitive advantage over the rest of the microbiota compared to baseline (Fig. 6F). These results highlight that both pathogens and potential pathobionts can specifically take advantage of the disrupted environment caused by bowel prep to invade beyond the intestinal lumen.

It is now well recognized that certain disruptions to the gut microbiota, such as antibiotic exposure, can dramatically impair colonization resistance and facilitate pathogen colonization. While antibiotics have long been understood as a critical factor in depleting the microbiota’s natural ability to prevent pathogen invasion, factors such as diet⁵⁰, cross-feeding interactions⁵¹, and microbiome diversity⁵² are now appreciated as important modulators of colonization resistance against enteric pathogens⁵³. In this study, we show that disruptions to the gut environment caused by bowel prep enable Salmonella colonization and systemic translocation of IBD pathobionts in a mouse model.

Our findings reveal that, similar to multi-day exposures to osmotic laxatives¹⁷, exposure to a short but high dose of laxative via bowel prep leads to an increase in intestinal osmolality, a decrease in SCFA concentration, and depletion of the intestinal mucus layer (Fig. 1). At the peak of bowel prep-induced diarrhea (6 hours post-procedure), the microbiota community exhibited a lower culturable density in several intestinal compartments and an increase in aerotolerant bacteria in the cecum but remained largely unchanged in terms of sequenced membership (Fig. 1G). Changes in membership did however occur later post bowel prep (Fig. 3C,D). This differs from long-term PEG exposure, which led to a higher density of culturable bacteria that was significantly altered in microbiota composition¹⁷. While long-term PEG exposure led to permanent changes in the microbiota due to the irreversible loss of specific members¹⁷, the original microbiota diversity was restored within days in our acute bowel prep model (Fig. 3C,D). This resilience is also paralleled in human data showing the recovery of the microbiota in healthy humans post-bowel prep^4,5. These results suggest that the microbiota may recover more easily from acute perturbations than from chronic ones, even if the acute perturbations are larger in magnitude.

Despite the commensal microbiota and gut environment being able to recover post-bowel prep, the gut environment does become vulnerable to pathogens during the window of disruption following treatment. Importantly, bowel prep enabled as few as 1,000 Salmonella to successfully colonize and expand in the gut as well as translocate to extraintestinal organs (Fig. 2G,H). Salmonella expansion occurred despite the absence of bicarbonate treatment in our model, which is commonly used to ensure the Salmonella load is not negatively affected by stomach acidity⁵⁴. These findings show that bowel prep renders the gut extremely susceptible to pathogen invasion and expansion. In light of this, additional measures to support intestinal resilience against infection post-bowel prep should be considered in bowel prep recovery strategies, particularly for at-risk populations. Future studies should explore strategies to enhance microbiota recovery and promote colonization resistance following bowel prep, such as modifications to bowel prep formulations that mitigate pathogen adherence and protect the exposed mucosal surface.

Our data suggests that a key factor in the increased sensitivity to pathogens post-bowel prep stems from the depletion of SCFA levels (Fig. 3E), as some colonization was possible when osmolality and mucus had recovered by 24 hours post-bowel prep (Fig. 3G,H). However, the highest levels of colonization are achieved at the height of bowel prep, when the intestinal osmolality, microbiota, SCFA and mucus layer were altered (Fig. 3). These findings are in line with previous work showing that the thinning of the intestinal mucus layer leads to enhanced bacterial penetration and susceptibility to pathogen infection^50,55. Importantly, mice genetically engineered to lack intestinal mucus (Muc2 -/-) display increased susceptibility to Citrobacter rodentium⁵⁶ as well as Salmonella³³ infection. Interestingly, despite mucus loss occurring across the digestive tract, Salmonella were found to preferentially infect cecal tissues and showed pathology distinct from that of streptomycin-treated mouse infection (Fig. 2E,F vs S2E,F), in which the mucus layer was not depleted⁵⁷.

Salmonella has been shown to require a functioning flagellum for effective colonization of the mammalian GI tract⁵⁴, but this requirement was circumvented following mucus layer depletion post-bowel prep (Fig. 4A-C). Specifically, we found that the non-motile mutant Salmonella ΔflhD was able to both colonize to similar levels as the wild-type and translocate to the mesenteric lymph nodes (Fig. 4A-C). Importantly, since FlhD is involved in coordinating virulence⁵⁸, the ability of Salmonella ΔflhD to translocate suggests that other virulence factors may facilitate Salmonella translocation post-bowel prep. This finding expands our understanding of pathogen-host interactions in disrupted gut environments and suggests that non-motile pathogens may also be able to exploit these conditions to invade the gut mucosa.

The diminished barrier to infection resulting from bowel prep raised the question of whether microbes already present in the endogenous microbiota could exploit changes in the gut environment, as observed in the case of an IBD microbiota. Supporting this notion, we found that pathobionts common in patients with IBD are highly resilient to the osmotic perturbation experienced during bowel prep (Fig. 6A), and our in vivo data suggest they can expand and translocate effectively in the post-bowel prep gut (Fig. 6D-F). Our data also show that healthy human or mouse microbiota do not have the same ability to reach (or be maintained in) extraintestinal organs post-bowel prep (Fig. 6D).

Differences in gut physiology and microbiota composition between humans and mice could play a major role in bowel prep outcomes; however, this research highlights the potential risks of bowel prep in vulnerable populations. Future research should focus on the effects of bowel prep on patients carrying pathobiont-rich microbiota. Due to the technical challenges of measuring bacterial translocation in humans and reported negative outcomes of patients with IBD following bowel preparation^11–16, this animal model may still provide important insights into which bacteria should be monitored in patients requiring more frequent colonoscopies. Beyond bowel prep, our findings suggest that bouts of osmotic diarrhea, such as those caused by malabsorption, could also promote pathobiont-driven negative health outcomes in people harboring these bacteria in their microbiota.

In conclusion, our study highlights the complex interplay that exists between the gut microbiota, mucus layer integrity, and pathogen colonization in the context of bowel prep. By elucidating these interactions, we can better understand the potential risks associated with depletion of the mucus layer and microbiota and develop more effective strategies to mitigate these effects.

Acknowledgements

The authors acknowledge that the land we performed this research on is the traditional, ancestral, and unceded territory of the xwməθkwəy̓əm (Musqueam) Nation. The land it is situated on has always been a place of learning for the Musqueam people, who for millennia have passed on in their culture, history, and traditions from one generation to the next on this site. We encourage others to learn more about the native lands in which they live and work at https://native-land.ca/.

The authors thank Denise Monack and members of the Tropini Lab for useful conversations and insight, in particular Michael Hunter and Jerry He for critically reading the manuscript and Angele Arrieta for ordering supplies and managing lab processes. We acknowledge the University of British Columbia Centre for Disease Modeling and Natalia Carranza Garcia for support with animal work. This work also received support from the Life Sciences Institute Biofactorial High-Throughput Biology Core, supported by the University of British Columbia Global Research Excellence Biological Resilience Initiative. We also thank Deng Wanyin and the Finlay lab for providing Salmonella strains.

The authors acknowledge support from: Canadian Institute for Advanced Research / Humans and the Microbiome (FL-001253 Appt 3362, to C.T.), Michael Smith Foundation for Health Research Scholar Award (18239, to C.T.), Canada Foundation for Innovation / Infrastructure Operating Fund (38277), Canada Tier 2 Research Chair, Quantitative Microbiota Biology for Health Applications (CRC-2022-00036, to C.T.), Canadian Institutes of Health Research (CIHR) Project grant (PJT 191743), Crohn's and Colitis Canada / Grants In Aid of Research (625155), Paul Allen Foundation / Allen Distinguished Investigators (ADI) Program (12935, to C.T. and S.R.S.) and the the CH.I.L.D Foundation Chair in Pediatric Gastroenterology (to B.A.V.).

CRediT authorship contribution statement:

CAC: Conceptualization, Methodology, Formal Analysis, Investigation, Data Curation, Writing – original draft, Writing - Review & Editing, Visualization

GM: Methodology, Formal Analysis, Investigation, Writing - Review & Editing, Visualization

KN: Conceptualization, Methodology, Investigation, Writing - Review & Editing, Supervision

AS: Investigation, Formal Analysis

DT: Investigation

CS: Investigation

IP: Formal Analysis

DMP: Investigation

TF: Investigation

SRS: Conceptualization, Resources, Supervision

MS: Resources, Supervision

BAV: Conceptualization, Resources

CT: Conceptualization, Resources, Writing – original draft, Writing– review & editing, Formal Analysis, Supervision, Project administration, Funding acquisition.

Declarations of interest: none

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Availability of data and materials

All the datasets generated and/or analyzed during the current study as well as the scripts used to plot the data are available in a Borealis repository:

https://borealisdata.ca/privateurl.xhtml?token=552f390a-f6d7-4b5c-aa52-055c5b9c5fab

Animal Handling and Ethics

All animal experiments were conducted in accordance with the ethical guidelines of the University of British Columbia’s (UBC) animal care procedures, following protocol number A23-0115 approved by the Animal Care Committee. Unless otherwise noted, C57BL/6J male and female mice between 8-12 weeks of age were used and were provided with an autoclaved standard diet (Purina LabDiet 5K67). As we did not observe sex differences, male and female mice were analyzed together in all experiments. Experimental groups that underwent oral gavage were monitored at 1 hour and 24 hours post-procedure. Daily health checks for mortality after infection were performed, and mice were euthanized if they lost more than 20% of their body weight or displayed signs of distress. Euthanasia at indicated tissue-collection time points was performed using carbon dioxide asphyxiation followed by cervical dislocation.

Bowel Preparation Mouse Model

Bowel prep solution was prepared by dissolving 42.5% (w/v) polyethylene glycol (PEG) (commercially branded Restoralax) in ddH₂O and filter sterilized. Food was withdrawn from all cages 1 hour before the bowel prep procedure. Mice were orally gavaged with 200 μL of bowel prep solution every 20 minutes a total of 4 times. Vehicle-treated mice were orally gavaged with 200 μL of water following the same dosing time as bowel prep-treated mice. Food and water were readministered after the procedure. Following bowel prep, we evaluated the impact of high dose osmotic laxatives on the gut environment and the gut microbiota at 6, 24, 48, and 72 hours post-bowel prep and compared to untreated controls sacrificed and the 6-hour timepoint (baseline).

Cecal Osmolality and pH Measurements

Mice were sacrificed, and cecal contents were collected and placed on ice. The osmolality of the cecal contents was directly measured using an Advanced Instruments Osmo1 Single-Sample Micro-Osmometer (Fisher Scientific, 22-047-531). pH was measured with a calibrated micro pH probe (Orion PerpHecT ROSS Combination pH Micro Electrode, Catalog number: 8220BNWP).

Serum and Tissue Collection

Serum was collected via cardiac puncture before tissue collection. Sterile tools were used to collect the lower lobe of the liver, mesenteric lymph nodes, and the spleen. The tissues were placed in pre-weighed sterile 2 mL tubes containing 200 μL of PBS and were put on ice. Cecal contents were collected, and half of the contents were flash-frozen on dry ice, while the other half was placed on ice for downstream analysis. The ileum, cecum, and colon tissues were collected in histology cassettes and immediately fixed in methacarn solution (60% dry methanol (Fisher Chemical, A412P-4), 30% chloroform (Fisher Chemical, C298-500), 10% glacial acetic acid (Fisher Chemical, A38-212)) for 7 to 14 days. The samples were then washed twice in methanol for 30 minutes, twice in 100% ethanol (Sigma, P016EAAN) for 20 minutes, and twice in xylenes (Fisher Chemical, X3P-1GAL) for 15 minutes. The tissues were coated and incubated in paraffin at 60°C for 2 hours and then dried at room temperature. Paraffin blocks were cut into 4 μm sections and mounted on slides by the British Columbia Children’s Hospital Research Institute’s Histology Core⁵⁹.

Lectin Staining of the GIT

Paraffin was removed from sectioned slides through incubation at 60^oC for 10 minutes, followed by 2 incubations in pre-warmed xylenes at 60^oC. Slides were then incubated in 99.5% ethanol for 5 minutes, then left to dry and circled with a PAP (liquid blocker) pen (Fisher Scientific)⁵⁹. DAPI (10 μg/mL, Sigma Aldrich), UEA-1 (Rhodamine-labeled Ulex Europaeus Agglutinin I, 40 μg/mL, Vector Laboratories), and WGA (Wheat Germ Agglutinin, 30 μg/mL, Vector Laboratories) were applied to fully cover the samples (approximately 250 μL) and incubated in the dark at room temperature for 45 minutes (Table 1). The slides were then washed in PBS 3 times. Sections were left to dry for 5 minutes, and ProLong Gold Antifade Mountant, (Invitrogen, P10144), was applied, followed by a #1.5 glass coverslip⁵⁹. Images were collected using a Zeiss LSM 900 confocal microscope with the ZEN 2020 software. Mucus thickness was quantified using the analysis platform BacSpace²⁸.

Table 1 Fluorescent Imaging Stains

Counter Stain	Conjugate Fluorophore	Supplier/ cat. #	Working concentration
DAPI	NA	Sigma Aldrich/ D9542-5MG	10 μg/mL
WGA	Rhodamine Red-X	Vector Laboratories / RL-1022	30 μg/mL
UEA-1	Fluorescein	Vector Laboratories / FL-1061	40 μg/mL

Confocal Image Straightening and Mucus Quantification

Confocal images of longitudinal sections of different organs (ileum, cecum, proximal and distal colon) were processed using the BacSpace image analysis pipeline using MATLAB software²⁸. Briefly, BacSpace identifies the epithelial boundary based on DAPI signal and straightens images relative to the epithelium. To quantify inner mucus layer thickness, the steepest decrease in WGA or UEA-1 signal between two user-defined coordinates along the x-axis of straightened images (corresponding to two distances from the epithelium) was identified at each position along the epithelium. Distance from this boundary to the epithelial edge was defined as thickness of the inner mucus layer at a given point along the epithelium. The average thickness of two sections from 3 different mice were calculated in every organ. To calculate mucus coverage, the ratio of epithelial segments with non-zero mucus layer values to those with detected mucus was calculated. Differences in average thickness between treatment and control groups (Fig. 1) or between timepoints post-bowel prep (Fig. 3) were assessed using a one-way ANOVA.

Hematoxylin & Eosin Tissue Histology

Paraffin was melted from the section slides by heating the slides in coplin jars at 60°C for 10 minutes. Slides were then incubated in the coplin jars in 60 ^oC xylenes for 3 minutes twice followed by 2 incubations in 100% EtOH for 2 minutes each, 95% EtOH for 2 minutes, and in ddH20 for 2 minutes⁶⁰. Next, the hematoxylin stain was performed by filling the coplin jars with Hematoxylin Solution, Gill’s No. 2 (Sigma Aldrich, GHS216), and incubating for 3 minutes, then in ddH₂O for 1 minute, differentiator solution (0.3% v/v 10N HCl [Fisher Chemical, SA49], 70% v/v EtOH [Sigma, P016EAAN] in ddH2O)) for 30 seconds, ddH20 for 1 minute, and blueing reagent (0.2% w/v NaHCO₃ [Fisher Chemical, BP328-500], 4.1% w/v MgSO₄ᐧ6H₂O [Fisher BioReagents, BP213-1] in ddH2O)) for 1 minute. For the Eosin stain, slides remained in the coplin jar and were filled with 95% EtOH for 1 minute followed by Eosin Y solution (Sigma-Aldrich, HTT110116), for 45 seconds. The stain was followed by washes in 95% EtOH for 1 minute, in 100% EtOH for 1 minute 3 times, and in xylenes for 2 minutes twice. Stained slides were mounted using Permount (Fisher Chemical, SP15-100), a xylene-based mounting medium, and allowed to solidify for at least 24 hours⁶⁰. H&E images were acquired using the 3D HISTECH Slide Scanner at 37x.

Pathology Scoring

H&E-stained sections of the cecum and distal colon were assessed for pathology in the lumen, epithelium, mucosa, and submucosa of the gut. Luminal pathology was scored based on the presence of necrotic epithelial cells and polymorphonuclear neutrophils (PMNs) (0 = none, 1 = scant, 2 = moderate, 3 = dense). The epithelium was scored for desquamation (0 = no change, 1 = limited shedding, 2 = moderate shedding per lesion), regenerative change (0 = none, 1 = mild, 2 = moderate, 3 = severe), ulceration (1 = epithelial ulceration), and PMNs in the epithelium (1 = PMNs present). The mucosa was scored based on crypt abscesses (0 = none, 1 = mild, 2 = moderate, 3 = severe) and the presence of mucin plugs and granulation tissue (1 = present). Lastly, the submucosa was scored for edema, mononuclear cell infiltration, and PMN infiltration (0 = no change, 1 = mild, 2 = moderate, 3 = severe). Scores for each location across the image were summed together, with the maximum pathological score being 24 per image. Images were scored by two blinded scorers, and the scores were averaged for each image.

Mucosa Thickness Measurements

20X images of H&E-stained tissues were exported in TIFF format from SlideViewer. TIFF images were converted from RGB to 8-bit format in FIJI, and pixel values were inverted so that tissues appeared light against a dark background. BacSpace was used to create two masks; one mask outlined the submucosal edge of the mucosa, and the other mask outlined the luminal edge of the epithelium. Both masks were applied to the original image so that only the mucosa and epithelium were non-zero values. BacSpace was then used on the masked images to straighten the image relative to the luminal edge of the epithelium. The luminal edge of the epithelium in the straightened image was detected as the greatest decrease in signal along the x-axis of the straightened image at each point along the epithelium. The mean thickness was quantified for each image, and the mean thicknesses of two images from the cecum and two images from the distal colon from three different mice per treatment (total = 6 images per treatment per organ) were compared using a Wilcoxon rank test.

Quantification of Bacterial Levels in Feces

1 μL of feces was diluted in 200 μL of sterile PBS. The solution was serially diluted at 1:10 down a 96-well plate. Next, 5 μL fecal dilutions were spot-plated on both LB-streptomycin agar plates and Columbia Blood agar plates. Plates were incubated aerobically overnight at 37 ^oC. Single colonies were counted from the highest possible dilution and back calculated to determine the absolute microbial abundance.

DNA Extraction, Library Preparation, 16S rRNA Sequencing and Analysis

DNA was extracted from fecal pellets and cecal contents using the DNeasy 96 PowerSoil Pro QIAcube HT Kit (Qiagen Inc., Valencia, CA) according to the manufacturer’s instructions.

For all 16S rRNA runs except for the in vitro IBD (Fig. 6B,C), 16S rRNA library preparation was conducted at the Biofactorial High-Throughput Biology (Bio!) Facility at the University of British Columbia. Amplification of the V4 region was performed with 515F/926R primers (515F, 5’- GTGYCAGCMGCCGCGGTAA-3’; 926R, 5’-CCGYCAATTYMTTTRAGTTT-3’). Pooled libraries were then submitted to the Bio! facility, where sequencing was performed on the Illumina MiSeq^TMplatform with v2 2 x 300 bp paired-end read chemistry. For the in vitro IBD samples, 16S library preparation was performed by Gut4Health as previously described⁶¹. Amplicons of the V4 region of the 16S rRNA were generated using KAPA HiFi HotStart Real-time PCR Master Mix (Roche) and barcode primers 515F: GTGYCAGCMGCCGCGGTAA and 806R: GGACTACNVGGGTWTCTAAT. Purified PCR libraries were normalized and pooled with the SequalPrepTM normalization plate (Applied Biosystems). Library concentrations were confirmed using the QubitTM dsDNA high sensitivity assay kit (Invitrogen) and KAPA Library Quantification Kit (Roche). The purified pooled libraries were then submitted to UBC’s Bioinformatics and Sequencing Consortium (SBC). Paired end read sequencing was carried out either on the Illumina MiSeqTM v3 platform with 2 x 300 bp paired end-read chemistry or the NextSeq 600 cycle P1 with 2 x 301 bp. To ensure DNA quality and quantity, an Agilent high sensitivity DNA kit (Agilent) was employed on an Agilent 2100 Bioanalyzer.

Read quality was assessed by running FASTQC⁶² on the generated FASTQ files. Reads were then imported into QIIME2-2023.9 for subsequent analyses⁶³. DADA2 (via q2-dada2) was used to denoise and quality filter the data; then reads were trimmed to remove primer sequences while maintaining mean Phred quality scores >Q30⁶⁴. Using the QIIME classification plugin (q2-feature-classifier), amplicon sequence variants (ASVs) were classified via a naïve Bayes machine-learning taxonomic classifier against the SILVA 138 99% identity reference sequence database⁶⁵. Multiple sequence alignment and phylogenetic tree generation was performed using MAFFT (via q2-alignment) and FastTree2 respectively (via q2-phylogeny)^66,67. Plotting was conducted using R v4.2.2⁶⁸. Tidyverse⁶⁹ and ggplot2⁷⁰ packages were used for data visualization. The packages phyloseq⁷¹, ggpubr⁷², and vegan⁷³ were used for sample rarefaction, calculation, and visualization of alpha and beta diversity metrics.

SCFA Extraction from Cecal Contents

SCFAs were extracted from 40 to 100 mg of flash-frozen cecal contents. The samples were homogenized with 0.8 mL of 25% phosphoric acid (LabChem) and centrifuged at 15,000 × g for 10 minutes at 4°C. The supernatant was removed from all samples and centrifuged again. Subsequently, 800 μL of the supernatant was filtered through a 0.45 µm filter (Fisher) and mixed with 0.2 mL of the internal standard solution containing 24.5 mmol/L isocaproic acid in a GC vial (12 × 32mm, Thermo Scientific). SCFAs were quantified by gas chromatography-mass spectrometry by the AFNS Chromatography Facility at the University of Alberta, as previously described⁷⁴.

Salmonella Culture and Inoculum Preparation

Salmonella ΔflhD was a gift from the Finlay Lab, UBC. Glycerol stocks of the naturally streptomycin resistant Salmonella enterica Typhimurium SL1344 strains (Salmonella (WT) and Salmonella ΔflhD) were streaked onto Luria-Bertani Miller (LB) 1.5% agar plates supplemented with streptomycin (100 µg/mL). Following incubation at 37°C for 24 hours without agitation, a single Salmonella colony was selected from the agar plate and transferred to 5 mL of LB liquid broth containing 100 µg/mL streptomycin. The culture was then aerobically incubated at 37°C with shaking at 200 rpm for 17 hours. Subsequently, the cultures were serially diluted in PBS to achieve the desired inoculant bacterial counts.

Salmonella Osmolality Growth Measurements

Wildtype Salmonella or Salmonella ΔflhD were diluted 1:100 from overnight LB cultures in fresh LB media adjusted to ~400, 800, 1200, and 1800 mOsm/Kg using PEG (Restoralax). Growth curves were obtained using a Biotek Synergy H1 plate reader at 37°C for 24 hours aerobically. The plates were shaken orbitally every 15 minutes following the collection of OD600 measurements. Background absorbance subtraction was performed from non-inoculated wells. The growth rates of the strains were determined using a previously published MATLAB package¹⁸.

Streptomycin Salmonella Infection Model

Conventional mice were fasted for 4 hours before being treated with 100 μL of 200 mg/mL streptomycin (Sigma-Aldrich, S6501-50G) by oral gavage³². 24 hours later, mice were fasted for 3 hours and infected by oral gavage with 100 μL of 10⁶ CFU Salmonella. Fecal samples were collected daily for 3 days after Salmonella infection and spot-plated on streptomycin-LB agar plates.

Bowel Preparation Salmonella Infection Model

Conventional mice underwent bowel prep as described previously and received oral gavage with 100 μL of 10⁶ CFU of wildtype Salmonella or Salmonella ΔflhD 6 hours after the bowel prep. To assess dose-dependent Salmonella colonization, mice were infected with 10², 10³, 10⁵, 10⁶, and 10⁹ CFU post-bowel prep. To explore the susceptibility to Salmonella infection following bowel prep over time, mice were infected with 10⁶ CFU of Salmonella 6, 24, and 48 hours post-bowel prep. Fecal samples were collected daily for 3 days after Salmonella infection and spot-plated on Columbia Blood agar plates (per liter: 35 g Columbia Broth, 5% Sheep Blood, hemin, and vitamin K) to determine total absolute bacterial abundance and streptomycin-LB agar plates to determine absolute abundance of Salmonella.

RNA extraction, Sequencing and Analysis

RNA was extracted from cecal tip tissue using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. The extracted RNA was sent to the Sequencing and Bioinformatics Consortium at UBC, where a sequencing library was prepared using a standard Illumina Stranded mRNA kit (Illumina) to generate 150 bp pair-end reads. Raw reads were assessed for nucleotide quality (terminal bases below PHRED quality score 20 were removed), trimmed to remove adaptor sequences, and merged using Fastp v. 0.23.4⁷⁵. The reads were then aligned to the mouse genome GRCm39 using the STAR 2.7 software⁷⁶ with --quantMode GeneCounts, and the gene counts obtained were imported into R for downstream analyses. Raw gene counts were analysed using DESeq2⁷⁷. For volcano plots, genes with adjusted p-value (Benjamini-Hochberg FDR corrected) less than 0.05 and Log₂ fold change greater than 1.5 were highlighted. For select genes of interest, the Z-scores of the variance stabilized counts calculated using DESeq2 were plotted for all timepoints. Data visualization was performed using the tidyverse⁶⁹, pheatmap⁷⁸, and ggplot2⁷⁰ packages in R.

Quantification of Bacterial Colonization in Extraintestinal Tissues

Liver, mesenteric lymph nodes, and spleen were collected in pre-weighed sterile tubes filled with 150 µL of PBS and weighed for final tissue mass. Tissues were homogenized at 300 Hz for 6 minutes using the TissueLyser II (QIAGEN). Homogenized samples were serially diluted at 1:20 in PBS, and all dilutions were spot-plated on both LB-streptomycin agar plates and Columbia Blood agar plates. Plates were incubated aerobically overnight at 37^oC. Single colonies were counted and back calculated to determine the absolute abundance of aerobic bacterial translocation to extraintestinal organs (mLN, spleen, and liver). Excess homogenized tissue was frozen at -80^oC for downstream 16S rRNA sequencing.

Pathobiont Growth Measurements

1 µL of glycerol stocks of each of the 130 pathobionts isolated from UC patients were streaked on LB agar plates. A single colony was inoculated into 5 mL of liquid LB-streptomycin broth and grown for 17 hours overnight aerobically and shaking at 200 rpm. All strains were subcultured 1:15 into LB broth in 2 mL 96 well plates, using a pipetting robot (INTEGRA VIAFLO384) and grown for 2 hours at 37^oC¹⁸. Strains were then inoculated at a ratio of 1:75 into LB media with pH levels of 4.0, 5.5, 6.9, and 8.0, and osmolality levels of 400, 800, 1200, and 1800 mOsm/Kg, adjusted with PEG. The experiments were conducted in technical triplicates using a 384-well plate.

Pathobiont bacterial growth curves were obtained using a Biotek Synergy H1 plate reader at 37°C for 24 hours either inside an anaerobic chamber (Coy Laboratories) with an atmosphere of 5% CO₂, 5% H₂, and 90% N2 (Linde Canada) or in aerobic conditions in parallel. The plates were shaken orbitally every 15 minutes following the collection of OD600 measurements. Background absorbance subtraction was performed from non-inoculated wells. The growth rates of the strains were determined using a previously published MATLAB package¹⁸.

Human Samples

All studies were approved by the Stanford University Institutional Review Board (IRB). Fecal samples were collected from patients with confirmed ulcerative colitis (UC) and from healthy controls. Stool samples were collected by patients at home in BIOME-Preserve tubes according to manufacturer instructions and returned to the research team within 24 hours, then snap-frozen with dry ice and stored at -80ºC until use. The fecal inocula for gavage were prepared in an anaerobic chamber, where 5g of feces from stool samples was resuspended in 10 mL of sterile and pre-reduced PBS and centrifuged at 4000 RPM for 5 minutes.

In vitro hIBD Microbiota Growth

2 g of human stool inocula were grown from the frozen fecal samples in 5 mL of MEGA medium^18,79 aerobically overnight and subcultured 1:200 in fresh medium at baseline or adjusted to 800 mOsm/Kg with PEG. 16S rRNA sequencing was performed as described above (DNA Extraction, Library Preparation, and 16S rRNA Sequencing).

Mouse Humanized Microbiota

Swiss Webster and C57BL/6J germ-free mice were inoculated with 200 µl of prepped fecal samples from human donors at 6 weeks of age by oral gavage (UC, hIBD or healthy control, hCTRL, respectively). In all experiments, offspring of inoculated mice between 8-12 weeks of age were used to ensure that mice were born with their respective microbiota. Stool was sampled approximately daily for two weeks (hIBD). Mice were sacrificed either before bowel prep (0 h), or at 24 hours or 2 weeks post-bowel prep and tissues were collected. Tissue was homogenized and plated as described above (Quantification of Bacterial Colonization in Extraintestinal Tissues). 16S rRNA sequencing from mesenteric lymph nodes was performed on pooled plated samples as described above (DNA Extraction, Library Preparation, and 16S rRNA Sequencing).

Statistics in Figures

For plating experiments where no CFUs were detected, the values were plotted at ½ the limit of detection (LoD) in log scale.

p > 0.05; ns (not significant), p < 0.05; *, p < 0.01; **, p < 0.001; ***, p < 0.0001; ****.

There is NO Competing Interest.

SIFigure1.pdf
Supplemental Figure 1. Bowel prep does not lead to overt pathology despite changes in cecal mass, mucus layer thickness and SCFA levels. A) The mass of the SI, cecum, and colon was measured in vehicle- and bowel prep- treated mice. B) Mucus coverage and mucus layer thickness quantification using Wheat Germ Agglutinin (WGA) lectin stain of the cecum and distal colon. C) Average pathological score of the distal colon and cecum in vehicle- and bowel prep- treated mice. D) Culturable bacterial density in the SI measured aerobically and anaerobically. E) SCFA abundance in the cecum in vehicle- and bowel prep- treated mice. F) Cecal pH of vehicle- and bowel prep-treated mice (A-E: Wilcoxon signed-rank test).
SIFigure2.pdf
Supplemental Figure 2. Comparison of bowel prep and streptomycin Salmonella models. A) Salmonella levels in feces in males vs females. B) Salmonella levels in the feces in bowel prep-treated mice (from Fig. 2C) compared to streptomycin-treated mice. C) Salmonella levels in feces were measured for 3 days after infection following streptomycin treatment by plating (Multiple Mann-Whitney U comparisons). D) Salmonella translocation to the extraintestinal organs 72 hours after inoculation. E) H&E-stained sections of the distal colon and cecum in vehicle and streptomycin- treated mice infected with 10⁶ CFU of Salmonella. F) Histopathological scoring of the distal colon and cecum in vehicle and streptomycin-treated mice inoculated with Salmonella. (A, B, C, D, F: Wilcoxon signed-rank test, LoD: Limit of Detection.).
SIFigure3.pdf
Supplemental Figure 3. Cecal mucus and SCFA measurements post-bowel prep in absence of Salmonella infection. A) (top) Representative confocal images of the cecum stained with DAPI and UEA-1 at baseline, 6, 24, and 48 hours post-bowel prep. Below are UEA-1 channel images straightened relative to the epithelium and highlighting the edge of the mucus layer detected and used in mucus coverage and thickness quantification. B) Cecal mucus thickness measured with UEA-1 stained mucus at baseline, 6, and 24 hours post-bowel prep. C) Cecal mucus coverage (left) and thickness (right) measured with WGA-stained mucus at baseline, 6, and 24 hours post-bowel prep. D) SCFA abundance at baseline, 6, 24, 48, and 72 hours post-bowel prep. (A, B: One-way ANOVA with Tukey multiple comparison).
SIFigure4.pdf
Supplemental Figure 4. Salmonella ΔflhD growth is robust in high osmolality conditions. A) Normalized maximum growth rate and B) normalized maximum optical density (OD600) of wild type Salmonella and Salmonella ΔflhD grown aerobically in media of varying osmolalities adjusted with PEG.
SIFigure5.jpg
Supplemental Figure 5. Bowel prep minimally impacts early gene expression but significantly alters circadian rhythm gene expression at 72 hours post-treatment. A) Differential gene expression analysis at 72 hours post-bowel prep highlighting significant changes in circadian rhythm-associated genes compared to baseline at 6 hours. Transcriptomics data was analysed with DESeq2 and adjusted p-value (Benjamini-Hochberg FDR corrected) less than 0.05 and Log₂ fold change greater than 1.5 are highlighted. B) Z-score for top 50 differentially expressed genes. Circadian rhythm genes Dbp, Tef, Per2-3, Ciart were all significantly downregulated, while Nfil3 and Bmal1 were upregulated compared to baseline. Dbp (D-box binding protein) is involved in the regulation of circadian gene expression and controls the transcription of several clock-controlled genes, including Tef (thyrotroph embryonic factor), which plays a role in circadian regulation⁴². Additionally, Per genes (including Per2 and Per3) are core components of the circadian clock that encode the PERIOD proteins, which usually accumulate in the active phase (night for mice^43,44) to help maintain the circadian rhythm^42,45. Nfil3 (nuclear factor interleukin 3-regulated) is known to repress Dbp while Bmal1 (Basic Helix-Loop-Helix ARNT Like 1) activates Per genes as part of the core transcriptional-translational feedback loop.
SIFigure6.pdf
Supplemental Figure 6. Aerobic growth of pathobionts in varying osmolality levels. A) Normalized maximum OD600 of 130 IBD-associated pathobiont strains under aerobic conditions at varying osmolality levels (400, 800, 1200, and 1800 mOsm/Kg) adjusted with NaCl. B) Schematic representation of the humanized mouse models (healthy and IBD). Germ-free mice were humanized with patient samples and were bred for experimental use. Offspring between 8-12 weeks of age were used for bowel prep experiments and samples were collected over 2 weeks.

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Bowel preparation-induced changes in the gut environment facilitate pathogen and pathobiont colonization

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Bowel prep rapidly alters the gut environment without immediate changes in host gene expression

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