Diet-driven differential response of Akkermansia muciniphila modulates pathogen susceptibility.

The erosion of the colonic mucus layer by a dietary fiber-deprived gut microbiota results in heightened susceptibility to an attaching and effacing pathogen, Citrobacter rodentium. Nevertheless, the questions of whether and how specific mucolytic bacteria aid in the increased pathogen susceptibility remain unexplored. Here, we leverage a functionally characterized, 14-member synthetic human microbiota in gnotobiotic mice to deduce which bacteria and functions are responsible for the pathogen susceptibility. Using strain dropouts of mucolytic bacteria from the community, we show that Akkermansia muciniphila renders the host more vulnerable to the mucosal pathogen during fiber deprivation. However, the presence of A. muciniphila reduces pathogen load on a fiber-sufficient diet, highlighting the context-dependent beneficial effects of this mucin specialist. The enhanced pathogen susceptibility is not owing to altered host immune or pathogen responses, but is driven by a combination of increased mucus penetrability and altered activities of A. muciniphila and other community members. Our study provides novel insights into the mechanisms of how discrete functional responses of the same mucolytic bacterium either resist or enhance enteric pathogen susceptibility.

Opposing diet, microbiome, and metabolite mechanisms regulate inflammatory bowel disease in a genetically susceptible host.

Inflammatory bowel diseases (IBDs) are chronic conditions characterized by periods of spontaneous intestinal inflammation and are increasing in industrialized populations. Combined with host genetics, diet and gut bacteria are thought to contribute prominently to IBDs, but mechanisms are still emerging. In mice lacking the IBD-associated cytokine, interleukin-10, we show that a fiber-deprived gut microbiota promotes the deterioration of colonic mucus, leading to lethal colitis. Inflammation starts with the expansion of natural killer cells and altered immunoglobulin-A coating of some bacteria. Lethal colitis is then driven by Th1 immune responses to increased activities of mucin-degrading bacteria that cause inflammation first in regions with thinner mucus. A fiber-free exclusive enteral nutrition diet also induces mucus erosion but inhibits inflammation by simultaneously increasing an anti-inflammatory bacterial metabolite, isobutyrate. Our findings underscore the importance of focusing on microbial functions-not taxa-contributing to IBDs and that some diet-mediated functions can oppose those that promote disease.

Unravelling specific diet and gut microbial contributions to inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a chronic condition characterized by periods of spontaneous intestinal inflammation and is increasing in industrialized populations. Combined with host genetic predisposition, diet and gut bacteria are thought to be prominent features contributing to IBD, but little is known about the precise mechanisms involved. Here, we show that low dietary fiber promotes bacterial erosion of protective colonic mucus, leading to lethal colitis in mice lacking the IBD-associated cytokine, interleukin-10. Diet-induced inflammation is driven by mucin-degrading bacteria-mediated Th1 immune responses and is preceded by expansion of natural killer T cells and reduced immunoglobulin A coating of some bacteria. Surprisingly, an exclusive enteral nutrition diet, also lacking dietary fiber, reduced disease by increasing bacterial production of isobutyrate, which is dependent on the presence of a specific bacterial species, Eubacterium rectale. Our results illuminate a mechanistic framework using gnotobiotic mice to unravel the complex web of diet, host and microbial factors that influence IBD.

A steamed broccoli sprout diet preparation that reduces colitis via the gut microbiota.

Sulforaphane is a bioactive metabolite with anti-inflammatory activity and is derived from the glucosinolate glucoraphanin, which is highly abundant in broccoli sprouts. However, due to its inherent instability its use as a therapeutic against inflammatory diseases has been limited. There are few studies to investigate a whole food approach to increase sulforaphane levels with therapeutic effect and reduce inflammation. In the current study, using a mouse model of inflammatory bowel disease, we investigated the ability of steamed broccoli sprouts to ameliorate colitis and the role of the gut microbiota in mediating any effects. We observed that despite inactivation of the plant myrosinase enzyme responsible for the generation of sulforaphane via steaming, measurable levels of sulforaphane were detectable in the colon tissue and feces of mice after ingestion of steamed broccoli sprouts. In addition, this preparation of broccoli sprouts was also capable of reducing chemically-induced colitis. This protective effect was dependent on the presence of an intact microbiota, highlighting an important role for the gut microbiota in the metabolism of cruciferous vegetables to generate bioactive metabolites and promote their anti-inflammatory effects.

Diet-driven differential response of Akkermansia muciniphila modulates pathogen susceptibility.

The erosion of the colonic mucus layer by a dietary fiber-deprived gut microbiota results in heightened susceptibility to an attaching and effacing pathogen, Citrobacter rodentium. Nevertheless, the questions of whether and how specific mucolytic bacteria aid in the increased pathogen susceptibility remain unexplored. Here, we leverage a functionally characterized, 14-member synthetic human microbiota in gnotobiotic mice to deduce which bacteria and functions are responsible for the pathogen susceptibility. Using strain dropouts of mucolytic bacteria from the community, we show that Akkermansia muciniphila renders the host more vulnerable to the mucosal pathogen during fiber deprivation. However, the presence of A. muciniphila reduces pathogen load on a fiber-sufficient diet, highlighting the context-dependent beneficial effects of this mucin specialist. The enhanced pathogen susceptibility is not owing to altered host immune or pathogen responses, but is driven by a combination of increased mucus penetrability and altered activities of A. muciniphila and other community members. Our study provides novel insights into the mechanisms of how discrete functional responses of the same mucolytic bacterium either resist or enhance enteric pathogen susceptibility.

Prototype endoscopic photoacoustic-ultrasound balloon catheter for characterizing intestinal obstruction.

In our previous studies, we have demonstrated the feasibility of characterizing intestinal inflammation and fibrosis using endoscopic photoacoustic imaging. Purposed at te clinical translation of the imaging technology, we developed a photoacoustic/ultrasound imaging probe by integrating a miniaturized ultrasound array and an angle-tipped optical fiber in a hydrostatic balloon catheter. When collapsed, the catheter probe may potentially be compatible with a clinical ileo-colonoscope. In addition, the flexible surface of the hydrostatic balloon allows for acoustic coupling at the uneven surfaces of the gas-filled intestine. Tissue phantom studies show that the catheter probe possesses an imaging penetration of at least 12 mm. Experiments with a rabbit model in vivo validated the probe in differentiating normal, acute and chronic conditions in intestinal obstruction.

Mcc1229, an Stx2a-Amplifying Microcin, Is Produced In Vivo and Requires CirA for Activity.

Enterohemorrhagic Escherichia coli (EHEC) strains, including the foodborne pathogen E. coli O157:H7, are responsible for thousands of hospitalizations each year. Various environmental triggers can modulate pathogenicity in EHEC by inducing the expression of Shiga toxin (Stx), which is encoded on a lambdoid prophage and transcribed together with phage late genes. Cell-free supernatants of the sequence type 73 (ST73) E. coli strain 0.1229 are potent inducers of Stx2a production in EHEC, suggesting that 0.1229 secretes a factor that activates the SOS response and leads to phage lysis. We previously demonstrated that this factor, designated microcin 1229 (Mcc1229), was proteinaceous and plasmid-encoded. To further characterize Mcc1229 and support its classification as a microcin, we investigated its regulation, determined its receptor, and identified loci providing immunity. The production of Mcc1229 was increased upon iron limitation, as determined by an enzyme-linked immunosorbent assay (ELISA), lacZ fusions, and quantitative real-time PCR (qRT-PCR). Spontaneous Mcc1229-resistant mutants and targeted gene deletion revealed that CirA was the Mcc1229 receptor. TonB, which interacts with CirA in the periplasm, was also essential for Mcc1229 import. Subcloning of the Mcc1229 plasmid indicated that Mcc activity was neutralized by two open reading frames (ORFs), each predicted to encode a domain of unknown function (DUF)-containing protein. In a germfree mouse model of infection, colonization with 0.1229 suppressed subsequent colonization by EHEC. Although Mcc1229 was produced in vivo, it was dispensable for colonization suppression. The regulation, import, and immunity determinants identified here are consistent with features of other Mccs, suggesting that Mcc1229 should be included in this class of small molecules.

Effect of ABT-263 on Intestinal Fibrosis in Human Myofibroblasts, Human Intestinal Organoids, and the Mouse Salmonella typhimurium Model.

BACKGROUND: Intestinal fibrosis and subsequent intestinal obstruction are common complications of Crohn's disease (CD). Current therapeutics combat inflammation, but no pharmacological therapy exists for fibrostenotic disease. Pathological persistence of activated intestinal myofibroblasts is a key driver of fibrosis in CD. In other organ systems, BH-3 mimetic drugs that affect Bcl-2 apoptotic pathways induce apoptosis in activated myofibroblasts and reduce fibrogenic gene expression, thereby reducing fibrosis. METHODS: We evaluated the proapoptotic and antifibrotic efficacy of several classes of BH-3 mimetics in 2 in vitro fibrogenesis models. The candidate molecule, ABT-263, was advanced to a 3-dimensional human intestinal organoid (HIO) model. Finally, the therapeutic efficacy of ABT-263 was evaluated in the mouse Salmonella typhimurium intestinal fibrosis model. RESULTS: The BH-3 mimetics induced apoptosis, repressed fibrotic protein expression, and reduced fibrogenic gene expression in normal human intestinal myofibroblasts. The BH-3 mimetics that target Bcl-2 and Bcl-xl demonstrated the greatest efficacy in vitro. The ABT-199 and ABT-263 induced apoptosis and ameliorated fibrogenesis in the in vitro myofibroblast models. In the HIO model, ABT-263 inhibited fibrogenesis and induced apoptosis. In the mouse S. typhimurium model, dose-dependent reduction in macroscopic pathology, histological inflammation, inflammatory and fibrotic gene expression, and extracellular matrix protein expression indicated ABT-263 may reduce intestinal fibrosis. CONCLUSIONS: In vitro, the antifibrotic efficacy of BH-3 mimetics identifies the Bcl-2 pathway as a druggable target and BH-3 mimetics as putative therapeutics. Reduction of inflammation and fibrosis in the mouse intestinal fibrosis model by ABT-263 indicates BH-3 mimetics as potential, novel antifibrotic therapeutics for Crohn's disease.

Intestinal fibrosis is a common complication of Crohn's disease, yet no effective therapies exist to treat fibrostenotic disease. We report ABT-263 (navitoclax) reduces intestinal fibrosis in in vitro models and reduces inflammation and fibrosis in a mouse IBD model.

Dendritic cell-derived TGF-ß mediates the induction of mucosal regulatory T-cell response to Helicobacter infection essential for maintenance of immune tolerance in mice.

BACKGROUND: Helicobacter pylori infection leads to regulatory T-cell (Treg) induction in infected mice, which contributes to H. pylori immune escape. However, the mechanisms responsible for H. pylori induction of Treg and immune tolerance remain unclear. We hypothesized DC-produced TGF-ß may be responsible for Treg induction and immune tolerance. MATERIALS AND METHODS: To test this hypothesis, we generated TGF-ß∆DC mice (CD11c+ DC-specific TGF-ß deletion) and assessed the impact of DC-specific TGF-ß deletion on DC function during Helicobacter infection in vitro and in vivo. To examine the T cell-independent DC function, we crossed TGF-ß∆DC mice onto Rag1KO background to generate TGF-ß∆DC xRag1KO mice. RESULTS: When stimulated with H. pylori, TGF-ß∆DC BMDC/splenocyte cocultures showed increased levels of proinflammatory cytokines and decreased levels of anti-inflammatory cytokines compared to control, indicating a proinflammatory DC phenotype. Following 6 months of H. felis infection, TGF-ß∆DC mice developed more severe gastritis and a trend toward more metaplasia compared to TGF-ßfl/fl with increased levels of inflammatory Th1 cytokine mRNA and lower gastric H. felis colonization compared to infected TGF-ßfl/fl mice. In a T cell-deficient background using TGF-ß∆DC xRag1KO mice, H. felis colonization was significantly lower when DC-derived TGF-ß was absent, revealing a direct, innate function of DC in controlling H. felis infection independent of Treg induction. CONCLUSIONS: Our findings indicate that DC-derived TGF-ß mediates Helicobacter-induced Treg response and attenuates the inflammatory Th1 response. We also demonstrated a previously unrecognized innate role of DC controlling Helicobacter colonization via a Treg-independent mechanism. DC TGF-ß signaling may represent an important target in the management of H. pylori.

The Gene Expression Profile of Uropathogenic Escherichia coli in Women with Uncomplicated Urinary Tract Infections Is Recapitulated in the Mouse Model.

Uropathogenic Escherichia coli (UPEC) is the primary causative agent of uncomplicated urinary tract infections (UTIs). UPEC fitness and virulence determinants have been evaluated in a variety of laboratory settings, including a well-established mouse model of UTI. However, the extent to which bacterial physiologies differ between experimental models and human infections remains largely understudied. To address this important issue, we compared the transcriptomes of three different UPEC isolates in human infection and under a variety of laboratory conditions, including LB culture, filter-sterilized urine culture, and the UTI mouse model. We observed high correlation in gene expression between the mouse model and human infection in all three strains examined (Pearson correlation coefficients of 0.86 to 0.87). Only 175 of 3,266 (5.4%) genes shared by all three strains had significantly different expression levels, with the majority of them (145 genes) downregulated in patients. Importantly, gene expression levels of both canonical virulence factors and metabolic machinery were highly similar between the mouse model and human infection, while the in vitro conditions displayed more substantial differences. Interestingly, comparison of gene expression between the mouse model and human infection hinted at differences in bladder oxygenation as well as nutrient composition. In summary, our work strongly validates the continued use of this mouse model for the study of the pathogenesis of human UTI.IMPORTANCE Different experimental models have been used to study UPEC pathogenesis, including in vitro cultures in different media, tissue culture, and mouse models of infection. The last is especially important since it allows evaluation of mechanisms of pathogenesis and potential therapeutic strategies against UPEC. Bacterial physiology is greatly shaped by environment, and it is therefore critical to understand how closely bacterial physiology in any experimental model relates to human infection. In this study, we found strong correlation in bacterial gene expression between the mouse model and human UTI using identical strains, suggesting that the mouse model accurately mimics human infection, definitively supporting its continued use in UTI research.

The Intermucosal Connection between the Mouth and Gut in Commensal Pathobiont-Driven Colitis.

The precise mechanism by which oral infection contributes to the pathogenesis of extra-oral diseases remains unclear. Here, we report that periodontal inflammation exacerbates gut inflammation in vivo. Periodontitis leads to expansion of oral pathobionts, including Klebsiella and Enterobacter species, in the oral cavity. Amassed oral pathobionts are ingested and translocate to the gut, where they activate the inflammasome in colonic mononuclear phagocytes, triggering inflammation. In parallel, periodontitis results in generation of oral pathobiont-reactive Th17 cells in the oral cavity. Oral pathobiont-reactive Th17 cells are imprinted with gut tropism and migrate to the inflamed gut. When in the gut, Th17 cells of oral origin can be activated by translocated oral pathobionts and cause development of colitis, but they are not activated by gut-resident microbes. Thus, oral inflammation, such as periodontitis, exacerbates gut inflammation by supplying the gut with both colitogenic pathobionts and pathogenic T cells.

Gut Microbiota Modulate CD8 T Cell Responses to Influence Colitis-Associated Tumorigenesis.

There is increasing evidence that gut microbiome perturbations, also known as dysbiosis, can influence colorectal cancer development. To understand the mechanisms by which the gut microbiome modulates cancer susceptibility, we examine two wild-type mouse colonies with distinct gut microbial communities that develop significantly different tumor numbers using a mouse model of inflammation-associated tumorigenesis. We demonstrate that adaptive immune cells contribute to the different tumor susceptibilities associated with the two microbial communities. Mice that develop more tumors have increased colon lamina propria CD8+ IFNγ+ T cells before tumorigenesis but reduced CD8+ IFNγ+ T cells in tumors and adjacent tissues compared with mice that develop fewer tumors. Notably, intratumoral T cells in mice that develop more tumors exhibit increased exhaustion. Thus, these studies suggest that microbial dysbiosis can contribute to colon tumor susceptibility by hyperstimulating CD8 T cells to promote chronic inflammation and early T cell exhaustion, which can reduce anti-tumor immunity.

Interleukin-22-mediated host glycosylation prevents Clostridioides difficile infection by modulating the metabolic activity of the gut microbiota.

The involvement of host immunity in the gut microbiota-mediated colonization resistance to Clostridioides difficile infection (CDI) is incompletely understood. Here, we show that interleukin (IL)-22, induced by colonization of the gut microbiota, is crucial for the prevention of CDI in human microbiota-associated (HMA) mice. IL-22 signaling in HMA mice regulated host glycosylation, which enabled the growth of succinate-consuming bacteria Phascolarctobacterium spp. within the gut microbiome. Phascolarctobacterium reduced the availability of luminal succinate, a crucial metabolite for the growth of C. difficile, and therefore prevented the growth of C. difficile. IL-22-mediated host N-glycosylation is likely impaired in patients with ulcerative colitis (UC) and renders UC-HMA mice more susceptible to CDI. Transplantation of healthy human-derived microbiota or Phascolarctobacterium reduced luminal succinate levels and restored colonization resistance in UC-HMA mice. IL-22-mediated host glycosylation thus fosters the growth of commensal bacteria that compete with C. difficile for the nutritional niche.

Dietary L-serine confers a competitive fitness advantage to Enterobacteriaceae in the inflamed gut.

Metabolic reprogramming is associated with the adaptation of host cells to the disease environment, such as inflammation and cancer. However, little is known about microbial metabolic reprogramming or the role it plays in regulating the fitness of commensal and pathogenic bacteria in the gut. Here, we report that intestinal inflammation reprograms the metabolic pathways of Enterobacteriaceae, such as Escherichia coli LF82, in the gut to adapt to the inflammatory environment. We found that E. coli LF82 shifts its metabolism to catabolize L-serine in the inflamed gut in order to maximize its growth potential. However, L-serine catabolism has a minimal effect on its fitness in the healthy gut. In fact, the absence of genes involved in L-serine utilization reduces the competitive fitness of E. coli LF82 and Citrobacter rodentium only during inflammation. The concentration of luminal L-serine is largely dependent on dietary intake. Accordingly, withholding amino acids from the diet markedly reduces their availability in the gut lumen. Hence, inflammation-induced blooms of E. coli LF82 are significantly blunted when amino acids-particularly L-serine-are removed from the diet. Thus, the ability to catabolize L-serine increases bacterial fitness and provides Enterobacteriaceae with a growth advantage against competitors in the inflamed gut.

A Rare Opportunist, Morganella morganii, Decreases Severity of Polymicrobial Catheter-Associated Urinary Tract Infection.

Catheter-associated urinary tract infections (CAUTIs) are common hospital-acquired infections and frequently polymicrobial, which complicates effective treatment. However, few studies experimentally address the consequences of polymicrobial interactions within the urinary tract, and the clinical significance of polymicrobial bacteriuria is not fully understood. Proteus mirabilis is one of the most common causes of monomicrobial and polymicrobial CAUTI and frequently cocolonizes with Enterococcus faecalis, Escherichia coli, Providencia stuartii, and Morganella morganiiP. mirabilis infections are particularly challenging due to its potent urease enzyme, which facilitates formation of struvite crystals, catheter encrustation, blockage, and formation of urinary stones. We previously determined that interactions between P. mirabilis and other uropathogens can enhance P. mirabilis urease activity, resulting in greater disease severity during experimental polymicrobial infection. Our present work reveals that M. morganii acts on P. mirabilis in a contact-independent manner to decrease urease activity. Furthermore, M. morganii actively prevents urease enhancement by E. faecalis, P. stuartii, and E. coli Importantly, these interactions translate to modulation of disease severity during experimental CAUTI, predominantly through a urease-dependent mechanism. Thus, products secreted by multiple bacterial species in the milieu of the catheterized urinary tract can directly impact prognosis.

Characterizing intestinal strictures of Crohn's disease in vivo by endoscopic photoacoustic imaging.

Crohn's disease (CD) is one type of inflammatory bowel disease where both inflammation and fibrosis cause the thickening of the bowel wall and development of the strictures. Accurate assessment of the strictures is critical for the management of CD because the fibrotic strictures must be removed surgically. In this study, a prototype capsule-shaped acoustic resolution photoacoustic (PA) endoscope, which can perform mulitwavelength side-view scanning, was developed to characterize the intestinal strictures of CD. The imaging performance of the probe was tested in phantom experiments and a rabbit trinitrobenzene sulfonic acid (TNBS) model with acute (inflammatory only) or chronic (mixed fibrotic and inflammatory) colitis in vivo. The motion artifacts due to intestinal peristalsis and the respiratory motion of the animals were compensated to improve image qualities. Quantitative molecular component images derived from multi-wavelength PA measurements of normal, acute and chronic intestinal strictures demonstrated statistically significant differences among the three groups that were confirmed by histopathology. A longitudinal study demonstrated the capability of the system in monitoring the development of fibrosis. The results suggest that the proposed novel, capsule-shaped acoustic resolution PA endoscope can be used to characterize fibrostenotic disease in vivo.

Regulation of Gastric Lgr5+ve Cell Homeostasis by Bone Morphogenetic Protein (BMP) Signaling and Inflammatory Stimuli.

BACKGROUND & AIMS: Gastric Leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) cells exert important functions during injury and homeostasis. Bone morphogenetic protein (BMP) signaling regulates gastric inflammation and epithelial homeostasis. We investigated if BMP signaling controls the fate of Lgr5+ve cells during inflammation. METHODS: The H+/K+-adenosine triphosphatase ß-subunit promoter was used to express the BMP inhibitor noggin (Nog) in the stomach (H+/K+-Nog mice). Inhibition of BMP signaling in Lgr5 cells was achieved by crossing Lgr5-EGFP-ires-CreERT2 (Lgr5-Cre) mice to mice with floxed alleles of BMP receptor 1A (Lgr5-Cre;Bmpr1aflox/flox mice). Lgr5/GFP+ve cells were isolated using flow cytometry. Lineage tracing studies were conducted by crossing Lgr5-Cre mice to mice that express Nog and tdTomato (Lgr5-Cre;H+/K+-Nog;Rosa26-tdTom). Infection with Helicobacter felis was used to induce inflammation. Morphology of the mucosa was analyzed by H&E staining. Distribution of H+/K+-adenosine triphosphatase-, IF-, Ki67-, CD44-, CD44v9-, and bromodeoxyuridine-positive cells was analyzed by immunostaining. Expression of neck and pit cell mucins was determined by staining with the lectins Griffonia (Bandeiraea) simplicifolia lectin II and Ulex europaeus agglutinin 1, respectively. Id1, Bmpr1a, Lgr5, c-Myc, and Cd44 messenger RNAs were measured by quantitative reverse-transcription polymerase chain reaction. RESULTS: Lgr5-Cre;Bmpr1aflox/flox mice showed diminished expression of Bmpr1a in Lgr5/GFP+ve cells. Infection of Lgr5-Cre;Bmpr1aflox/flox mice with H felis led to enhanced inflammation, increased cell proliferation, parietal cell loss, and to the development of metaplasia and dysplasia. Infected Lgr5-Cre;H+/K+-Nog;Rosa26-tdTom mice, but not control mice, showed the presence of tomato+ve glands lining the lesser curvature that stained positively with Griffonia (Bandeiraea) simplicifolia lectin II and Ulex europaeus agglutinin 1, and with anti-IF, -CD44, -CD44v9, and -bromodeoxyuridine antibodies. CONCLUSIONS: Inflammation and inhibition of BMP signaling activate Lgr5+ve cells, which give rise to metaplastic, dysplastic, proliferating lineages that express markers of mucus neck and zymogenic cell differentiation.

Reduced infectivity of waterborne viable but nonculturable Helicobacter pylori strain SS1 in mice.

BACKGROUND: Helicobacter pylori infection has been consistently associated with lack of access to clean water and proper sanitation, but no studies have demonstrated that the transmission of viable but nonculturable (VBNC) H. pylori can occur from drinking contaminated water. In this study, we used a laboratory mouse model to test whether waterborne VBNCH. pylori could cause gastric infection. MATERIALS AND METHODS: We performed five mouse experiments to assess the infectivity of VBNCH. pylori in various exposure scenarios. VBNC viability was examined using Live/Dead staining and Biolog phenotype metabolism arrays. High doses of VBNCH. pylori in water were chosen to test the "worst-case" scenario for different periods of time. One experiment also investigated the infectious capabilities of VBNC SS1 using gavage. Further, immunocompromised mice were exposed to examine infectivity among potentially vulnerable groups. After exposure, mice were euthanized and their stomachs were examined for H. pylori infection using culture and PCR methodology. RESULTS: VBNC cells were membrane intact and retained metabolic activity. Mice exposed to VBNCH. pylori via drinking water and gavage were not infected, despite the various exposure scenarios (immunocompromised, high doses) that might have permitted infection with VBNCH. pylori. The positive controls exposed to viable, culturable H. pylori did become infected. CONCLUSIONS: While other studies that have used viable, culturable SS1 via gavage or drinking water exposures to successfully infect mice, in our study, waterborne VBNC SS1 failed to colonize mice under all test conditions. Future studies could examine different H. pylori strains in similar exposure scenarios to compare the relative infectivity of the VBNC vs the viable, culturable state, which would help inform future risk assessments of H. pylori in water.