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Not all patients with cancer and severe neutropenia develop fever, and the fecal microbiome may play a role. In a single-center study of patients undergoing hematopoietic cell transplant ( n  = 119), the fecal microbiome was characterized at onset of severe neutropenia. A total of 63 patients (53%) developed a subsequent fever, and their fecal microbiome displayed increased relative abundances of Akkermansia muciniphila , a species of mucin-degrading bacteria ( P  = 0.006, corrected for multiple comparisons). Two therapies that induce neutropenia, irradiation and melphalan, similarly expanded A. muciniphila and additionally thinned the colonic mucus layer in mice. Caloric restriction of unirradiated mice also expanded A. muciniphila and thinned the colonic mucus layer. Antibiotic treatment to eradicate A. muciniphila before caloric restriction preserved colonic mucus, whereas A. muciniphila reintroduction restored mucus thinning. Caloric restriction of unirradiated mice raised colonic luminal pH and reduced acetate, propionate, and butyrate. Culturing A. muciniphila in vitro with propionate reduced utilization of mucin as well as of fucose. Treating irradiated mice with an antibiotic targeting A. muciniphila or propionate preserved the mucus layer, suppressed translocation of flagellin, reduced inflammatory cytokines in the colon, and improved thermoregulation. These results suggest that diet, metabolites, and colonic mucus link the microbiome to neutropenic fever and may guide future microbiome-based preventive strategies.

Immune checkpoint inhibitors (ICIs) target advanced malignancies with high efficacy but also predispose patients to immune-related adverse events like immune-mediated colitis (IMC). Given the association between gut bacteria with response to ICI therapy and subsequent IMC, fecal microbiota transplantation (FMT) represents a feasible way to manipulate microbial composition in patients, with a potential benefit for IMC. Here, we present a large case series of 12 patients with refractory IMC who underwent FMT from healthy donors as salvage therapy. All 12 patients had grade 3 or 4 ICI-related diarrhea or colitis that failed to respond to standard first-line (corticosteroids) and second-line immunosuppression (infliximab or vedolizumab). Ten patients (83%) achieved symptom improvement after FMT, and three patients (25%) required repeat FMT, two of whom had no subsequent response. At the end of the study, 92% achieved IMC clinical remission. 16 S rRNA sequencing of patient stool samples revealed that compositional differences between FMT donors and patients with IMC before FMT were associated with a complete response after FMT. Comparison of pre- and post-FMT stool samples in patients with complete responses showed significant increases in alpha diversity and increases in the abundances of Collinsella and Bifidobacterium , which were depleted in FMT responders before FMT. Histologically evaluable complete response patients also had decreases in select immune cells , including CD8 + T cells, in the colon after FMT when compared with non-complete response patients ( n = 4). This study validates FMT as an effective treatment strategy for IMC and gives insights into the microbial signatures that may play a critical role in FMT response.

Fever in the setting of low white blood cell counts, also known as neutropenic fever, is considered an oncologic emergency. Only some 30% of patients with neutropenic fever have an identifiable source of infection, such as a bloodstream bacterial infection of intestinal origin. Whether other nonpathogenic intestinal bacteria can contribute to neutropenic fever is not known. In a cohort of 119 patients undergoing hematopoietic cell transplantation, a treatment with a high risk for neutropenic fever, the fecal microbiome was examined at onset of neutropenia. Over the next 4 days, 63 patients (53%) developed a fever, which was associated with increased Akkermansia muciniphila, a species of intestinal commensal bacteria with mucus-degrading capabilities (p=0.006, corrected for multiple comparisons). In mouse models, two cytotoxic therapies, irradiation and melphalan, each also produced an expansion of fecal A. muciniphila as well as thinning of the colonic mucus layer. Direct irradiation of fecal bacteria, however, did not lead to expansion of A. muciniphila, suggesting an indirect effect via the host. Irradiated mice displayed reduced oral food intake, and dietary restriction of unirradiated mice was sufficient to produce an expansion of A. muciniphila and thinning of the colonic mucus layer. Treatment of diet-restricted mice with several narrow-spectrum antibiotics demonstrated that azithromycin, which depleted intestinal A. muciniphila, led to preservation of the colonic mucus layer. Diet-restricted mice developed an increase in colonic luminal pH and reductions in acetate, propionate, and butyrate. Treatment of A. muciniphila in vitro with lower pH and increased propionate led to delayed growth and prevented utilization of mucin. Dietary restriction and low propionate also produced large changes in A. muciniphila gene expression, including upregulation of L-fucose isomerase, a member of glycosyl hydrolase family 109, and a member of the Idh/MocA family of oxidoreductases, which may play roles in mucin glycan utilization. Supplementing the drinking water of diet-restricted mice with propionate led to preservation of the colonic mucus layer. In the setting of irradiation, we found that by 6 days following treatment mice developed signs of systemic infection manifesting as hypothermia. Upon examination of colon tissues, we found that irradiated mice had elevated levels of inflammatory cytokines including IL-1b, CCL2, CCL7, IL-22, CXCL1, and CXCL10. Treatment with azithromycin or propionate lessened the severity of hypothermia, preserved the mucus layer, and mitigated elevations in inflammatory cytokines in the colon. In summary, we have found that clinical neutropenic fever is associated with increased intestinal abundance of mucolytic bacteria, and further experiments in mice have identified a pathway linking impaired diet and loss of bacterial metabolites as important mediators of this process.

Patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT) are at risk for graft-versus-host disease (GVHD). The gastrointestinal tract has been identified to be a primary target of allogeneic donor T cells in allo-HSCT. The intestinal microbiota is known to interact with the host immune system and has been found to be an important modulator of GVHD. Broad-spectrum antibiotics such as carbapenems are often used in allo-HSCT patients to treat infections but have been found to increase the risk for intestinal GVHD, possibly via bystander depletion of beneficial commensal bacteria. However, whether loss of beneficial commensal bacteria is mechanistically sufficient to aggravate intestinal GVHD is not known. To examine this further, we utilized a mouse model of GVHD to study the effects of meropenem, a commonly used carbapenem in allo-HSCT patients. Lethally irradiated B6D2F1 (H-2 b/d) mice were intravenously injected with 5 × 10 6 bone marrow cells and 5 × 10 6 splenocytes from major histocompatibility complex-mismatched B6 (H-2 b) or syngeneic donors on day 0. Meropenem was additionally administered to some allo-HSCT recipient mice in the drinking water from days 3 to 15 after allo-HSCT. We found that meropenem treatment aggravated GVHD primarily in the colon of mice, and 16S ribosomal RNA gene sequencing analysis demonstrated that many bacterial genera were depleted in meropenem-treated mice, including Blautia and Lachnoclostridium that belong to the class Clostridia. Simultaneously, the abundance of the genus Bacteroides was increased in meropenem-treated mice, which we also observed in allo-HSCT patients at our institution following meropenem treatment. Interestingly, when meropenem-treated mice were additionally treated orally with decontaminating antibiotics, GVHD severity and survival were ameliorated, suggesting that meropenem could induce worsened GVHD via expansion of pro-inflammatory bacteria as well as depletion of beneficial bacteria. We explored which species of Bacteroides was the most expanded by meropenem treatment and identified Bacteroides thetaiotaomicron (BT) as significantly increased species. Reintroduction of BT restored aggravated GVHD in mice treated with decontaminating antibiotics. BT is a gram-negative obligate anaerobe with a broad ability to degrade dietary polysaccharides as well as host mucin glycans. In meropenem-treated allogeneic mice, we observed significantly thinner colonic mucus, increased myeloid-cell infiltration into colonic tissue, and increased bacterial translocation into mesenteric lymph nodes. Decontamination, in contrast, led to preservation of the colonic mucus layer in meropenem-treated allogeneic mice. BT, in the presence of multiple suitable carbohydrate substrates, has been found to preferentially consume certain carbohydrates first and only after depleting these will it then upregulate utilization genes targeting other available polysaccharides. Host mucin glycans are particularly low on the metabolic hierarchy for BT. RNA sequencing of stool samples from meropenem-treated allogeneic mice demonstrated that BT upregulated expression of β-galactosidase, sialidase and α-L-fucosidase, all of which participate in the degradation of host mucin glycans. Carbohydrate mass spectrometry profiling of colonic luminal contents showed decreased levels of polysaccharides comprised of xylose. Interestingly, xylose supplementation in meropenem-treated allogeneic mice significantly prevented thinning of the colonic mucus layer, indicating that providing an alternative carbohydrate source was sufficient to suppress mucus-degrading behavior in BT (Figure). In conclusion, expansion of BT in meropenem-treated allogeneic mice is strongly associated with GVHD-related mortality. Broad-spectrum antibiotics such as meropenem are useful for treating infections in allo-HSCT, but can also lead to an altered intestinal environment with changes in levels of microbial-derived metabolites or decreased nutrients. This, in turn, can lead to expansion and altered behavior of commensal bacteria such as BT to target intestinal mucus, which can contribute to increased severity of GVHD. Specific nutritional supplementation strategies such as providing xylose may be helpful to combat changes to the intestinal environment in allo-HSCT patients with antibiotic-mediated microbiome injury. Figure 1 Figure 1. Disclosures Jenq: MaaT Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; LisCure: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seres: Consultancy, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Kaleido: Consultancy, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Research Funding; Prolacta: Consultancy, Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy; Microbiome DX: Consultancy; Karius: Consultancy.

Acute gastrointestinal intestinal GVHD (aGI-GVHD) is a serious complication of allogeneic hematopoietic stem cell transplantation, and the intestinal microbiota is known to impact on its severity. However, an association between treatment response of aGI-GVHD and the intestinal microbiota has not been well-studied. In a cohort of patients with aGI-GVHD (n=37), we found that non-response to standard therapy with corticosteroids was associated with prior treatment with carbapenem antibiotics and loss of Bacteroides ovatus from the microbiome. In a mouse model of carbapenem-aggravated GVHD, introducing Bacteroides ovatus reduced severity of GVHD and improved survival. Bacteroides ovatus reduced degradation of colonic mucus by another intestinal commensal, Bacteroides thetaiotaomicron, via its ability to metabolize dietary polysaccharides into monosaccharides, which then inhibit mucus degradation by Bacteroides thetaiotaomicron and reduce GVHD-related mortality.