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Treatment with celecoxib, a selective COX-2 inhibitor, reduces formation of premalignant adenomatous polyps in the gastrointestinal tracts of humans and mice. In addition to its chemopreventive activity, celecoxib can exhibit antimicrobial activity. Differing bacterial profiles have been found in feces from colon cancer patients compared with those of normal subjects. Moreover, preclinical studies suggest that bacteria can modulate intestinal tumorigenesis by secreting specific metabolites. In the current study, we determined whether celecoxib treatment altered the luminal microbiota and metabolome in association with reducing intestinal polyp burden in mice. Administration of celecoxib for 10 weeks markedly reduced intestinal polyp burden in APCMin/+ mice. Treatment with celecoxib also altered select luminal bacterial populations in both APCMin/+ and wild-type mice, including decreased Lactobacillaceae and Bifidobacteriaceae as well as increased Coriobacteriaceae. Metabolomic analysis demonstrated that celecoxib caused a strong reduction in many fecal metabolites linked to carcinogenesis, including glucose, amino acids, nucleotides, and lipids. Ingenuity Pathway Analysis suggested that these changes in metabolites may contribute to reduced cell proliferation. To this end, we showed that celecoxib reduced cell proliferation in the base of normal appearing ileal and colonic crypts of APCMin/+ mice. Consistent with this finding, lineage tracing indicated that celecoxib treatment reduced the rate at which Lgr5-positive stem cells gave rise to differentiated cell types in the crypts. Taken together, these results demonstrate that celecoxib alters the luminal microbiota and metabolome along with reducing epithelial cell proliferation in mice. We hypothesize that these actions contribute to its chemopreventive activity. Cancer Prev Res; 9(9); 721–31. ©2016 AACR.

Pathway alterations associated with celecoxib-induced metabolite changes in WT mice.

Supplementary Figure S1. Administration of celecoxib alters the luminal metabolome of WT mice. Feces were collected at baseline (0 weeks) then again at 5 and 10 weeks after initiating celecoxib treatment.

Data are rank transformed and displayed as color intensity with low levels indicated by green color and high levels indicated by red color.

Supplementary Figure Legends.

PD-1 blockade (nivolumab) efficacy remains modest for metastatic sarcoma. In this paper, we present an open-label, non-randomized, non-comparative pilot study of bempegaldesleukin, a CD122-preferential interleukin-2 pathway agonist, with nivolumab in refractory sarcoma at Memorial Sloan Kettering/MD Anderson Cancer Centers (NCT03282344). We report on the primary outcome of objective response rate (ORR) and secondary endpoints of toxicity, clinical benefit, progression-free survival, overall survival, and durations of response/treatment. In 84 patients in 9 histotype cohorts, all patients experienced ≥1 adverse event and treatment-related adverse event; 1 death was possibly treatment-related. ORR was highest in angiosarcoma (3/8) and undifferentiated pleomorphic sarcoma (2/10), meeting predefined endpoints. Results of our exploratory investigation of predictive biomarkers show: CD8 + T cell infiltrates and PD-1 expression correlate with improved ORR; upregulation of immune-related pathways correlate with improved efficacy; Hedgehog pathway expression correlate with resistance. Exploration of this combination in selected sarcomas, and of Hedgehog signaling as a predictive biomarker, warrants further study in larger cohorts.

Patients with multiple myeloma (MM) who achieve minimal residual disease (MRD) negativity after upfront treatment have superior outcomes compared with those who remain MRD+. Recently, associations have been shown between specific commensal microbes and development of plasma cell disorders. Here, we report the association between intestinal microbiota composition and treatment outcome in MM. Microbiota composition of fecal samples collected from 34 MM patients after induction therapy and at the time of flow cytometry–based bone marrow MRD testing was determined by 16S ribosomal RNA sequencing. We observed a higher relative abundance of Eubacterium hallii in the 16 MRD− patients relative to the 18 MRD+ patients. No association was observed between microbial relative abundance and autologous stem cell transplantation history or MM paraprotein isotype. No differences in microbiota α diversity were observed between MRD− and MRD+ patients. The potential association of microbiota composition with treatment response in MM patients is an important parameter for additional correlative and clinical investigation.

Posttransplant diarrhea is associated with kidney allograft failure and death, but its etiology remains unknown in the majority of cases. Because altered gut microbial ecology is a potential basis for diarrhea, we investigated whether posttransplant diarrhea is associated with gut dysbiosis. We enrolled 71 kidney allograft recipients for serial fecal specimen collections in the first 3 months of transplantation and profiled the gut microbiota using 16S ribosomal RNA (rRNA) gene V4-V5 deep sequencing. The Shannon diversity index was significantly lower in 28 diarrheal fecal specimens from 25 recipients with posttransplant diarrhea than in 112 fecal specimens from 46 recipients without posttransplant diarrhea. We found a lower relative abundance of 13 commensal genera (Benjamini-Hochberg adjusted P ≤ .15) in the diarrheal fecal specimens including the same 4 genera identified in our prior study. The 28 diarrheal fecal specimens were also evaluated by a multiplexed polymerase chain reaction (PCR) assay for 22 bacterial, viral, and protozoan gastrointestinal pathogens, and 26 specimens were negative for infectious etiologies. Using PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) to predict metagenomic functions, we found that diarrheal fecal specimens had a lower abundance of metabolic genes. Our findings suggest that posttransplant diarrhea is not associated with common infectious diarrheal pathogens but with a gut dysbiosis.

PURPOSE Although primary germ cell tumors (GCTs) have been extensively characterized, molecular analysis of metastatic sites has been limited. We performed whole-exome sequencing and targeted next-generation sequencing on paired primary and metastatic GCT samples in a patient cohort enriched for cisplatin-resistant disease. PATIENTS AND METHODS Tissue sequencing was performed on 100 tumor specimens from 50 patients with metastatic GCT, and sequencing of plasma cell-free DNA was performed for a subset of patients. RESULTS The mutational landscape of primary and metastatic pairs from GCT patients was highly discordant (68% of all somatic mutations were discordant). Whereas genome duplication was common and highly concordant between primary and metastatic samples, only 25% of primary-metastasis pairs had ≥ 50% concordance at the level of DNA copy number alterations (CNAs). Evolutionary-based analyses revealed that most mutations arose after CNAs at the respective loci in both primary and metastatic samples, with oncogenic mutations enriched in the set of early-occurring mutations versus variants of unknown significance (VUSs). TP53 pathway alterations were identified in nine cisplatin-resistant patients and had the highest degree of concordance in primary and metastatic specimens, consistent with their association with this treatment-resistant phenotype. CONCLUSION Analysis of paired primary and metastatic GCT specimens revealed significant molecular heterogeneity for both CNAs and somatic mutations. Among loci demonstrating serial genetic evolution, most somatic mutations arose after CNAs, but oncogenic mutations were enriched in the set of early-occurring mutations as compared with VUSs. Alterations in TP53 were clonal when present and shared among primary-metastasis pairs.

The origin of most bacterial infections in the urinary tract is often presumed to be the gut. Herein, we investigate the relationship between the gut microbiota and future development of bacteriuria and urinary tract infection (UTI). We perform gut microbial profiling using 16S rRNA gene deep sequencing on 510 fecal specimens from 168 kidney transplant recipients and metagenomic sequencing on a subset of fecal specimens and urine supernatant specimens. We report that a 1% relative gut abundance of Escherichia is an independent risk factor for Escherichia bacteriuria and UTI and a 1% relative gut abundance of Enterococcus is an independent risk factor for Enterococcus bacteriuria. Strain analysis establishes a close strain level alignment between species found in the gut and in the urine in the same subjects. Our results support a gut microbiota–UTI axis, suggesting that modulating the gut microbiota may be a potential novel strategy to prevent UTIs., Urinary tract infections (UTIs) are associated with changes in the gut microbiome. Here, the authors evaluate the relationship between the gut microbiome and development of UTI in kidney transplant patients and show that uropathogenic gut abundance might represent a risk factor for development of bacteriuria and UTI.

Poor oral health appears to be a risk factor for pancreatic cancer, possibly implicating the oral microbiota. In this pilot study, we evaluated the characteristics of the oral microbiota in patients with pancreatic ductal adenocarcinoma (PDAC), intraductal papillary mucinous neoplasms (IPMN), and healthy controls. Forty newly diagnosed PDAC patients, 39 IPMN patients, and 58 controls, excluding current smokers and users of antibiotics, provided saliva samples. Common oral bacterial species were comprehensively surveyed by sequencing of the 16S rRNA microbial genes. We obtained measures of diversity and the mean relative proportions of individual taxa. We explored the degree to which these measures differed according to respondent characteristics based on individual interviews. PDAC cases did not differ in diversity measures from either controls or IPMN cases. PDAC cases had higher mean relative proportions of Firmicutes and related taxa, while controls had higher mean relative proportions of Proteobacteria and related taxa. Results were generally similar when comparing PDAC to IPMN cases. Among IPMNs and controls combined, younger individuals had higher levels of several taxa within the Proteobacteria. The only other variable consistently related to mean relative proportions was mouthwash use, with taxa within Firmicutes more common among users. While there were no differences in diversity of the oral microbiota among these groups, there were differences in the mean relative proportions of some taxa. Characteristics of the oral microbiota are not associated with most measures of oral health.

Purpose The major causes of mortality after allogeneic hematopoietic-cell transplantation (allo-HCT) are relapse, graft-versus-host disease (GVHD), and infection. We have reported previously that alterations in the intestinal flora are associated with GVHD, bacteremia, and reduced overall survival after allo-HCT. Because intestinal bacteria are potent modulators of systemic immune responses, including antitumor effects, we hypothesized that components of the intestinal flora could be associated with relapse after allo-HCT. Methods The intestinal microbiota of 541 patients admitted for allo-HCT was profiled by means of 16S ribosomal sequencing of prospectively collected stool samples. We examined the relationship between abundance of microbiota species or groups of related species and relapse/progression of disease during 2 years of follow-up time after allo-HCT by using cause-specific proportional hazards in a retrospective discovery-validation cohort study. Results Higher abundance of a bacterial group composed mostly of Eubacterium limosum in the validation set was associated with a decreased risk of relapse/progression of disease (hazard ratio [HR], 0.82 per 10-fold increase in abundance; 95% CI, 0.71 to 0.95; P = .009). When the patients were categorized according to presence or absence of this bacterial group, presence also was associated with less relapse/progression of disease (HR, 0.52; 95% CI, 0.31 to 0.87; P = .01). The 2-year cumulative incidences of relapse/progression among patients with and without this group of bacteria were 19.8% and 33.8%, respectively. These associations remained significant in multivariable models and were strongest among recipients of T-cell–replete allografts. Conclusion We found associations between the abundance of a group of bacteria in the intestinal flora and relapse/progression of disease after allo-HCT. These might serve as potential biomarkers or therapeutic targets to prevent relapse and improve survival after allo-HCT.

Klebsiella pneumoniae is a common cause of bloodstream infections in immunocompromised and hospitalized patients, and over the last 2 decades, some strains have acquired resistance to nearly all available antibiotics, including broad-spectrum carbapenems. The U.S. Centers for Disease Control and Prevention has listed carbapenem-resistant K. pneumoniae (CR-Kp) as an urgent public health threat. Dense colonization of the intestine by CR-Kp and other antibiotic-resistant bacteria is associated with an increased risk of bacteremia. Reducing the density of gut colonization by CR-Kp is likely to reduce their transmission from patient to patient in health care facilities as well as systemic infections. How CR-Kp expands and persists in the gut lumen, however, is poorly understood. Herein, we generated a highly saturated mutant library in a multidrug-resistant K. pneumoniae strain and identified genetic factors that are associated with dense gut colonization by K. pneumoniae. This study sheds light on host colonization by K. pneumoniae and identifies potential colonization factors that contribute to high-density persistence of K. pneumoniae in the intestine., A diverse, antibiotic-naive microbiota prevents highly antibiotic-resistant microbes, including carbapenem-resistant Klebsiella pneumoniae (CR-Kp), from achieving dense colonization of the intestinal lumen. Antibiotic-mediated destruction of the microbiota leads to expansion of CR-Kp in the gut, markedly increasing the risk of bacteremia in vulnerable patients. While preventing dense colonization represents a rational approach to reduce intra- and interpatient dissemination of CR-Kp, little is known about pathogen-associated factors that enable dense growth and persistence in the intestinal lumen. To identify genetic factors essential for dense colonization of the gut by CR-Kp, we constructed a highly saturated transposon mutant library with >150,000 unique mutations in an ST258 strain of CR-Kp and screened for in vitro growth and in vivo intestinal colonization in antibiotic-treated mice. Stochastic and partially reversible fluctuations in the representation of different mutations during dense colonization revealed the dynamic nature of intestinal microbial populations. We identified genes that are crucial for early and late stages of dense gut colonization and confirmed their role by testing isogenic mutants in in vivo competition assays with wild-type CR-Kp. Screening of the transposon library also identified mutations that enhanced in vivo CR-Kp growth. These newly identified colonization factors may provide novel therapeutic opportunities to reduce intestinal colonization by CR-Kp.

4058 Background: Pembrolizumab and trastuzumab (P&T) and chemotherapy demonstrated 27 month mOS, 13 month mPFS, and 91% response rate in first-line HER2-positive mEG cancer irrespective of PD-L1 status (Janjigian Lancet Oncology 2020). Biomarkers including 89Zr-trastuzumab PET, blood, and tumor analysis were correlated with progression-free survival. Methods: Twenty-five patients received P&T once 3 weeks prior to addition of chemotherapy to P&T. Pre-treatment tumor biopsies, 89Zr-trastuzumab PET scans, serial plasma ctDNA (Guardant360, Redwood City, CA) and CT scans were performed. Tumor-matched DNA alterations were identified by correlating ctDNA and tissue-NGS variant calls. Pre-, on-, and post-treatment biopsies were analyzed using WES and IHC (HER2, PD-L1). Biomarkers were correlated with mPFS and 6-month PFS, the primary endpoint. Results: Of patients with tumor-matched mutations ctDNA at baseline, 12 of 16 had a decline in their maxVAF by week 3, corresponding to a mPFS of 14.7 (11.0-NR) vs 5.9 (95% CI 4.1-NR) months (p=0.009) and a mOS of 29.7 (95% CI 27.2-NR) vs 7.71 (95% CI 6.6-NR) months (p=0.006). 9 of 12 (75%) patients with decline in ctDNA at 3 weeks post-P&T achieved the 6-month PFS primary endpoint while the 4 patients with no decline in ctDNA all progressed in under 6-months. Similarly, 7 of 9 (78%) patients who had a decline in CT-measurements in all disease sites achieved the 6 month PFS primary endpoint, versus 10 of 16 (62.5%) of patients who did not respond in all sites (p=0.66), suggesting that ctDNA is superior to CT as an early predictive biomarker of response. Lack of ERBB2 amplification (amp) by NGS in ctDNA and/or tumor was associated with lack of response to P&T alone prior to addition of chemotherapy. Interestingly, no lesions from patients lacking ERBB2 ctDNA amp (n=3) responded to induction P&T by CT, while lesions from 3/9 patients lacking ERBB2 tissue amp responded to P&T by 3-week CT, suggesting intrapatient HER2 heterogeneity. Eight patients also underwent 89Zr-Trastuzumab PET scans prior to P&T and up to 5 lesions per disease site were measured on serial CT scans. All 15 lesions with intense uptake (SUVmax>10) responded to P&T, but only 9/24 lesions with SUVmax89Zr-trastuzumab PET avidity are non-invasive predictive biomarkers of response to HER2-directed therapy. Evaluation of tumor immune environment digital spatial profiling is underway. Clinical trial information: NCT02954536.

The composition of the intestinal microbiota influences the development of inflammatory disorders. However, associating inflammatory diseases with specific microbial members of the microbiota is challenging, because clinically detectable inflammation and its treatment can alter the microbiota’s composition. Immunologic checkpoint blockade with ipilimumab, a monoclonal antibody that blocks cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) signalling, is associated with new-onset, immune-mediated colitis. Here we conduct a prospective study of patients with metastatic melanoma undergoing ipilimumab treatment and correlate the pre-inflammation faecal microbiota and microbiome composition with subsequent colitis development. We demonstrate that increased representation of bacteria belonging to the Bacteroidetes phylum is correlated with resistance to the development of checkpoint-blockade-induced colitis. Furthermore, a paucity of genetic pathways involved in polyamine transport and B vitamin biosynthesis is associated with an increased risk of colitis. Identification of these biomarkers may enable interventions to reduce the risk of inflammatory complications following cancer immunotherapy.

4559 Background: Pembrolizumab can be safely combined with trastuzumab and chemotherapy and has promising activity with median OS 27 months and 91% objective response rate in HER2-positive mEG cancer irrespective of PD-L1 status (NCT02954536; Janjigian ESMO 2019). Tumor biopsies and blood samples were collected in this phase II trial to identify molecular and immune predictors of response and resistance to PTC. Methods: Pre-treatment and post-progression biopsies were analyzed using WES and IHC (HER2, PD-L1). Peripheral blood was collected pre-treatment, every 9 weeks on-treatment and at progression for plasma ctDNA (Guardant 360, Guardant Health, Redwood, CA). Tumor-matched DNA alterations were identified by correlating ctDNA and solid tumor WES results. Landmark PFS analysis was used to compare ctDNA clearance status at 9 weeks post-treatment. Results: Baseline ctDNA was analysed from 31 of 37 patients of whom 84% (26/31) had tumor-matched ctDNA detected at baseline. Patients who cleared ctDNA at 9 weeks (n = 17/23) achieved a longer median PFS than those who did not - mPFS 12.3 months (95% CI 7.44-NA vs 3.9 months (95% CI 2.01-NA) (log-rank p = 0.02). On serial blood monitoring of 16 patients with eventual radiographic progression, ctDNA re-appearance preceded CT detection in 8 (50%) patients. WES was completed in 31 patients with pre-treatment, and 12 patients post-progression, including matched samples from 10 patients. Loss of HER2 over-expression/amplification was noted in 44% (7/16) of post-progression samples by IHC/FISH (2 IHC 0/1, 5 FISH-). In paired post-progression samples on WES, we observed loss of ERBB2 in 2 patients, and new amplifications of CCND1/3, FGF3/4/19, CDK6/12, KRAS, MYC, and MET, as well as mutations in KRAS, PIK3CD and PIK3RA. Plasma analysis at progression demonstrated copy number increases and/or new amplifications in MET, CKD6, PIK3CA, KRAS, FGFR2, EGFR and CCDN1 as well as KRAS, RB1, PTEN, NF1, NOTCH1, BRAF, and FGFR1 mutations. Conclusions: The majority of patients with previously untreated HER2 positive mEG have detectable plasma ctDNA at baseline. The re-appearance of ctDNA during therapy may serve as an early predictor of progression and help identify genetic drivers of acquired resistance. Loss of ERBB2 over-expression/amplification and activating MAPK alterations occur at PTC progression. Evaluation of tumor immune environment by multiplex IHC and additional ctDNA analysis is underway.

J Justin Mulvey,1,2 Eric R Littmann,3 Lilan Ling,3 Michael R McDevitt,4,5 Eric G Pamer,3,5 David A Scheinberg1,3,5 1Department of Laboratory Medicine, Weill Cornell Medicine, New York, NY, USA; 2Department of Molecular Molecular Pharmacology, Memorial Sloan Kettering Cancer Center, New York, NY, USA; 3Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA; 4Department of Radiology, Memorial Sloan Kettering Cancer Center, New York, NY, USA; 5Weill Cornell Medicine, New York, NY, USA Background: Amine-modified carbon nanotubes are drug delivery platforms with great potential that have not yet been applied in human clinical trials. Although modified nanotube vectors have the ability to carry multiple effectors, targeting agents, and even wrapped RNA, reports on unmodified, insoluble carbon nanotubes have highlighted inflammation in organs, including the intestine, with disruption of its resident microbiota. Disruption of the microbiota may allow for colonization by pathogenic bacteria, such as Clostridoidies difficile, stimulate immunoinfiltrates into the lamina propria or alter the absorption of therapeutics. Most proposed nanotube drugs are soluble, modified structures that are administered parenterally, and the majority of these soluble macromolecules are renally excreted; however, some are released into the bile, gaining access to the gastrointestinal tract. Methods: Using environmentally isolated BALB/C mice in oral and intraperitoneal dosing models, high dose (3.80 or 4.25 mg/week), we administered amine-modified, soluble carbon nanotubes for 7 or 8 weeks. The general health and weight of the mice were monitored weekly, and upon killing, the diversity and content of their colonic, cecal, and ileal microbiota were assessed using shotgun 16S DNA sequencing. Results and conclusion: We show that while oral administration at suprapharmacological doses modestly altered the α- and β-diversity of the mouse microbiome, these changes did not result in observed changes in clinical end points. Intraperitoneally-dosed mice exhibited none of the toxicities assessed. Keywords: SWCNT, toxicity, 16S sequencing, nanopharmaceuticals

Respiratory viral infections are frequent in patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HCT) and can potentially progress to lower respiratory tract infection (LRTI). The intestinal microbiota contributes to resistance against viral and bacterial pathogens in the lung. However, whether intestinal microbiota composition and associated changes in microbe-derived metabolites contribute to the risk of LRTI following upper respiratory tract viral infection remains unexplored in the setting of allo-HCT. Fecal samples from 360 allo-HCT patients were collected at the time of stem cell engraftment and subjected to deep, 16S ribosomal RNA gene sequencing to determine microbiota composition, and short-chain fatty acid levels were determined in a nested subset of fecal samples. The development of respiratory viral infections and LRTI was determined for 180 days following allo-HCT. Clinical and microbiota risk factors for LRTI were subsequently evaluated using survival analysis. Respiratory viral infection occurred in 149 (41.4%) patients. Of those, 47 (31.5%) developed LRTI. Patients with higher abundances of butyrate-producing bacteria were fivefold less likely to develop viral LRTI, independent of other factors (adjusted hazard ratio = 0.22, 95% confidence interval 0.04-0.69). Higher representation of butyrate-producing bacteria in the fecal microbiota is associated with increased resistance against respiratory viral infection with LRTI in allo-HCT patients.

The relationship between intestinal microbiota composition and acute graft-versus-host disease (GVHD) after allogeneic blood/marrow transplantation (allo BMT) is not well understood. Intestinal bacteria have long been thought to contribute to GVHD pathophysiology, but recent animal studies in non-transplant settings have found that anti-inflammatory effects are mediated by certain subpopulations of intestinal commensals. Hypothesizing that a more nuanced relationship may exist between the intestinal bacteria and GVHD, we evaluated the fecal bacterial composition of 64 patients 12 days after BMT. We found that increased bacterial diversity was associated with reduced GVHD-related mortality. Furthermore, harboring increased amounts of bacteria belonging to the genus Blautia was associated with reduced GVHD lethality in this cohort and was confirmed in another independent cohort of 51 patients from the same institution. Blautia abundance was also associated with improved overall survival. We evaluated the abundance of Blautia with respect to clinical factors and found that loss of Blautia was associated with: 1) treatment with antibiotics that inhibit anaerobic bacteria and 2) receiving total parenteral nutrition (TPN) for longer durations. We conclude that increased abundance of commensal bacteria belonging to the Blautia genus is associated with reduced lethal GVHD and improved overall survival.

Antibiotic treatment can deplete the commensal bacteria of a patient’s gut microbiota and, paradoxically, increase their risk of subsequent infections. In allogeneic hematopoietic stem cell transplantation (allo-HSCT), antibiotic administration is essential for optimal clinical outcomes but significantly disrupts intestinal microbiota diversity, leading to loss of many beneficial microbes. Although gut microbiota diversity loss during allo-HSCT is associated with increased mortality, approaches to reestablish depleted commensal bacteria have yet to be developed. We have initiated a randomized, controlled clinical trial of autologous fecal microbiota transplantation (auto-FMT) versus no intervention and have analyzed the intestinal microbiota profiles of 25 allo-HSCT patients (14 who received auto-FMT treatment and 11 control patients who did not). Changes in gut microbiota diversity and composition revealed that the auto-FMT intervention boosted microbial diversity and reestablished the intestinal microbiota composition that the patient had before antibiotic treatment and allo-HSCT. These results demonstrate the potential for fecal sample banking and posttreatment remediation of a patient’s gut microbiota after microbiota-depleting antibiotic treatment during allo-HSCT.

Clostridium difficile is a spore-forming anaerobic bacterium that causes colitis in patients with disrupted colonic microbiota. While some individuals are asymptomatic C. difficile carriers, symptomatic disease ranges from mild diarrhea to potentially lethal toxic megacolon. The wide disease spectrum has been attributed to the infected host’s age, underlying diseases, immune status, and microbiome composition. However, strain-specific differences in C. difficile virulence have also been implicated in determining colitis severity. Because patients infected with C. difficile are unique in terms of medical history, microbiome composition, and immune competence, determining the relative contribution of C. difficile virulence to disease severity has been challenging, and conclusions regarding the virulence of specific strains have been inconsistent. To address this, we used a mouse model to test 33 clinical C. difficile strains isolated from patients with disease severities ranging from asymptomatic carriage to severe colitis, and we determined their relative in vivo virulence in genetically identical, antibiotic-pretreated mice. We found that murine infections with C. difficile clade 2 strains (including multilocus sequence type 1/ribotype 027) were associated with higher lethality and that C. difficile strains associated with greater human disease severity caused more severe disease in mice. While toxin production was not strongly correlated with in vivo colonic pathology, the ability of C. difficile strains to grow in the presence of secondary bile acids was associated with greater disease severity. Whole-genome sequencing and identification of core and accessory genes identified a subset of accessory genes that distinguish high-virulence from lower-virulence C. difficile strains. IMPORTANCE Clostridium difficile is an important cause of hospital-associated intestinal infections, and recent years have seen an increase in the number and severity of cases in the United States. A patient’s antibiotic history, immune status, and medical comorbidities determine, in part, the severity of C. difficile infection. The relative virulence of different clinical C. difficile strains, although postulated to determine disease severity in patients, has been more difficult to consistently associate with mild versus severe colitis. We tested 33 distinct clinical C. difficile isolates for their ability to cause disease in genetically identical mice and found that C. difficile strains belonging to clade 2 were associated with higher mortality. Differences in survival were not attributed to differences in toxin production but likely resulted from the distinct gene content in the various clinical isolates.

Background Clostridium difficile infection (CDI) is a frequent complication in recipients of allogeneic hematopoietic stem cell transplantation (allo-HSCT), who receive intensive treatments that significantly disrupt the intestinal microbiota. In this study, we examined the microbiota composition of allo-HSCT recipients to identify bacterial colonizers that confer protection against CDI after engraftment. Methods Feces collected from adult recipients allo-HSCT at engraftment were analyzed; 16S ribosomal RNA genes were sequenced and analyzed from each sample. Bacterial taxa with protective effects against development of CDI were identified by means of linear discriminant analysis effect size analysis and then further assessed with clinical predictors of CDI using survival analysis. Results A total of 234 allo-HSCT recipients were studied; postengraftment CDI developed in 53 (22.6%). Within the composition of the microbiota, the presence of 3 distinct bacterial taxa was correlated with protection against CDI: Bacteroidetes, Lachnospiraceae, and Ruminococcaceae. Colonization with these groups at engraftment was associated with a 60% lower risk of CDI, independent of clinical factors. Conclusions Colonization with these 3 bacterial groups is associated with a lower risk of CDI. These groups have been shown to be vital components of the intestinal microbiota. Targeted efforts to maintain them may help minimize the risk of CDI in this at-risk population.

Becattini et al. provide evidence that a diverse gut microbiota antagonizes the foodborne pathogen Listeria monocytogenes in the intestinal lumen, thereby reducing bloodstream invasion. Microbiota perturbation by antibiotic treatment increases susceptibility to listeriosis, with dramatic effects in immunocompromised hosts., Listeria monocytogenes is a foodborne pathogen that causes septicemia, meningitis and chorioamnionitis and is associated with high mortality. Immunocompetent humans and animals, however, can tolerate high doses of L. monocytogenes without developing systemic disease. The intestinal microbiota provides colonization resistance against many orally acquired pathogens, and antibiotic-mediated depletion of the microbiota reduces host resistance to infection. Here we show that a diverse microbiota markedly reduces Listeria monocytogenes colonization of the gut lumen and prevents systemic dissemination. Antibiotic administration to mice before low dose oral inoculation increases L. monocytogenes growth in the intestine. In immunodeficient or chemotherapy-treated mice, the intestinal microbiota provides nonredundant defense against lethal, disseminated infection. We have assembled a consortium of commensal bacteria belonging to the Clostridiales order, which exerts in vitro antilisterial activity and confers in vivo resistance upon transfer into germ free mice. Thus, we demonstrate a defensive role of the gut microbiota against Listeria monocytogenes infection and identify intestinal commensal species that, by enhancing resistance against this pathogen, represent potential probiotics.

Listeria monocytogenes is a foodborne pathogen that causes septicemia, meningitis and chorioamnionitis and is associated with high mortality. Immunocompetent humans and animals, however, can tolerate high doses of L. monocytogenes without developing systemic disease. The intestinal microbiota provides colonization resistance against many orally acquired pathogens, and antibiotic-mediated depletion of the microbiota reduces host resistance to infection. Here we show that a diverse microbiota markedly reduces Listeria monocytogenes colonization of the gut lumen and prevents systemic dissemination. Antibiotic administration to mice before low dose oral inoculation increases L. monocytogenes growth in the intestine. In immunodeficient or chemotherapy-treated mice, the intestinal microbiota provides nonredundant defense against lethal, disseminated infection. We have assembled a consortium of commensal bacteria belonging to the Clostridiales order, which exerts in vitro antilisterial activity and confers in vivo resistance upon transfer into germ free mice. Thus, we demonstrate a defensive role of the gut microbiota against Listeria monocytogenes infection and identify intestinal commensal species that, by enhancing resistance against this pathogen, represent potential probiotics.

The gastrointestinal tracts of mammals are colonized by hundreds of microbial species that contribute to health, including colonization resistance against intestinal pathogens. Many antibiotics destroy intestinal microbial communities and increase susceptibility to intestinal pathogens. Among these, Clostridium difficile, a major cause of antibiotic-induced diarrhoea, greatly increases morbidity and mortality in hospitalized patients. Which intestinal bacteria provide resistance to C. difficile infection and their in vivo inhibitory mechanisms remain unclear. Here we correlate loss of specific bacterial taxa with development of infection, by treating mice with different antibiotics that result in distinct microbiota changes and lead to varied susceptibility to C. difficile. Mathematical modelling augmented by analyses of the microbiota of hospitalized patients identifies resistance-associated bacteria common to mice and humans. Using these platforms, we determine that Clostridium scindens, a bile acid 7α-dehydroxylating intestinal bacterium, is associated with resistance to C. difficile infection and, upon administration, enhances resistance to infection in a secondary bile acid dependent fashion. Using a workflow involving mouse models, clinical studies, metagenomic analyses, and mathematical modelling, we identify a probiotic candidate that corrects a clinically relevant microbiome deficiency. These findings have implications for the rational design of targeted antimicrobials as well as microbiome-based diagnostics and therapeutics for individuals at risk of C. difficile infection.

Antibiotic-mediated microbiota destruction and the consequent loss of colonization resistance can result in intestinal domination with vancomycin-resistant Enterococcus (VRE), leading to bloodstream infection in hospitalized patients. Clearance of VRE remains a challenging goal that, if achieved, would reduce systemic VRE infections and patient-to-patient transmission. Although obligate anaerobic commensal bacteria have been associated with colonization resistance to VRE, the specific bacterial species involved remain undefined. Herein, we demonstrate that a precisely defined consortium of commensal bacteria containing the Clostridium cluster XIVa species Blautia producta and Clostridium bolteae restores colonization resistance against VRE and clears VRE from the intestines of mice. While C. bolteae did not directly mediate VRE clearance, it enabled intestinal colonization with B. producta, which directly inhibited VRE growth. These findings suggest that therapeutic or prophylactic administration of defined bacterial consortia to individuals with compromised microbiota composition may reduce inter-patient transmission and intra-patient dissemination of highly antibiotic-resistant pathogens.

We report the complete genome sequence of a vancomycin-resistant isolate of Enterococcus faecium derived from human feces. The genome comprises one chromosome of 2.9 Mb and three plasmids. The strain harbors a plasmid-borne vanA- type vancomycin resistance locus and is a member of multilocus sequencing type (MLST) cluster ST-17.

Treatment with celecoxib, a selective COX-2 inhibitor, reduces formation of premalignant adenomatous polyps in the gastrointestinal tracts of humans and mice. In addition to its chemopreventive activity, celecoxib can exhibit antimicrobial activity. Differing bacterial profiles have been found in feces from colon cancer patients compared with those of normal subjects. Moreover, preclinical studies suggest that bacteria can modulate intestinal tumorigenesis by secreting specific metabolites. In the current study, we determined whether celecoxib treatment altered the luminal microbiota and metabolome in association with reducing intestinal polyp burden in mice. Administration of celecoxib for 10 weeks markedly reduced intestinal polyp burden in APCMin/+ mice. Treatment with celecoxib also altered select luminal bacterial populations in both APCMin/+ and wild-type mice, including decreased Lactobacillaceae and Bifidobacteriaceae as well as increased Coriobacteriaceae. Metabolomic analysis demonstrated that celecoxib caused a strong reduction in many fecal metabolites linked to carcinogenesis, including glucose, amino acids, nucleotides, and lipids. Ingenuity Pathway Analysis suggested that these changes in metabolites may contribute to reduced cell proliferation. To this end, we showed that celecoxib reduced cell proliferation in the base of normal appearing ileal and colonic crypts of APCMin/+ mice. Consistent with this finding, lineage tracing indicated that celecoxib treatment reduced the rate at which Lgr5-positive stem cells gave rise to differentiated cell types in the crypts. Taken together, these results demonstrate that celecoxib alters the luminal microbiota and metabolome along with reducing epithelial cell proliferation in mice. We hypothesize that these actions contribute to its chemopreventive activity. Cancer Prev Res; 9(9); 721–31. ©2016 AACR.

Background: Multiple myeloma (MM) patients who achieve minimal residual disease (MRD) negative status after upfront treatment have prolonged progression-free and overall survival compared with those who remain MRD(+) (Landgren CO, Devlin SM et al. Bone Marrow Transplant. 2016;51(12):1565-8). Commensal intestinal microbial composition has been associated with treatment outcomes in cancer patients. We sought to evaluate whether the composition of the intestinal microbiota is associated with MRD status in patients with MM. Methods: Stool samples were collected prospectively from 34 patients after completion of upfront therapy for MM at the time of MRD testing. MRD was assessed with next-generation flow cytometry of bone marrow aspirates (sensitivity 10-5). Microbial analysis was performed via sequencing of 16S rRNA V4-V5 regions using the Illumina MiSeq platform and sequence data was analyzed using UPARSE (Edgar RC, Nature Methods 2013;10(10):996-8). The linear discriminant effect size method (LEfSe) (Segata N et al. Genome Biol. 2011;12(6):R60.) was used to compare detected clades among all groups and evaluate for associations with outcomes, using MRD as class and autologous stem cell transplant (ASCT) as subclass. Alpha diversity was calculated by the Inverse Simpson index and differential relative abundance were calculated using the phyloseq package and compared using the Wilcoxon rank sum test on the R statistical computing platform. Results: Among 34 patients evaluable for microbiota composition and MRD status, the median age was 62.5 years and 16 (47.1%) were MRD(-) at time of stool collection. 24 (70.6%) were treated with carfilzomib, lenalidomide, and dexamethasone as induction therapy (MRD(-): 14 (87.5%), MRD(+):10 (55.5%). 4 (28.5%) MRD(-) patients had autologous stem cell transplant(ASCT), compared with 10 (55.5%) who were MRD(+). In the cohort's samples, we observed 19 phyla, 315 genera, 654 species, and 1549 operational taxonomic units (OTUs). There was no significant difference in alpha diversity between MRD(-) (median 12.24, IQR = 8.76-13.98) and MRD(+) patients (median 12.44, IQR = 8.36 -16.23), p=0.6 by Wilcoxon rank sum test. A positive association with MRD negativity was noted with two butyrate-producing organisms, Eubacterium hallii (p=0.001) and Faecalibacterium prausnitzii (p= 0.006). To further evaluate these relationships, we performed a differential abundance analysis of these selected taxa in MRD(+) and MRD(-) patients at the genus and species level. The relative abundance of the genera Eubacterium and Faecalibacterium were higher in fecal samples from MRD(-) patients than MRD(+) patients (Eubacterium MRD(-): median 4.51% (IQR = 2.83 - 7.32%) vs. MRD(+): median 3.07% (IQR = 1.35 - 3.87%), p=0.0326; Faecalibacterium MRD(-): median 1.68% (IQR = 0.69 - 7.48%) vs. MRD(+): median 0.003% (IQR = 0 - 3.19%), p=0.022. The relative abundance of both species of interest were higher in MRD(-) patients than in MRD(+) patients: E. hallii MRD(-): median 2.67% (IQR = 2.11 - 3.98%) vs. MRD(+): median 1.01% (IQR = 0 - 2.16%), p=0.001; F. prausnitzii MRD(-): median 1.43% (IQR = 0.53 - 7.28%) vs. MRD(+): median 0.3%, (IQR = 0 - 2.54%), p=0.022. Other species of Eubacterium and Faecalibacterium were not significantly differentially abundant between the two groups. Conclusions: Intestinal microbiota containing several butyrate-producing anaerobes appear to be associated with MRD-negativity in patients with myeloma, with higher relative abundance of Eubacterium hallii and Faecalibacterium prausnitzii in MRD(-) patients compared with MRD(+) patients. Butyrate and other short-chain fatty acids are biologically active metabolites formed during microbial fermentation of dietary or host-derived carbohydrates, which supply the host with energy and also modulate immunity, including exerting anti-inflammatory functions. Microbes of the genus Eubacterium have been associated with reduced risk of relapse in several hematologic cancers after allogeneic hematopoietic cell transplantation, including MM (Peled JU, Devlin SM et al. J Clin Oncol 2017;35(15):1650-9). This is first study to our knowledge to suggest an association between gut microbiota and MRD status in patients with myeloma and supports further investigation of a potential role for intestinal microbiota in the natural history and treatment of myeloma. Disclosures Peled: Seres Therapeutics: Research Funding. Landgren:Karyopharm: Consultancy; Merck: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Lesokhin:Squibb: Consultancy, Honoraria; Genentech: Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding; Serametrix, inc.: Patents & Royalties: Royalties; Takeda: Consultancy, Honoraria; Janssen: Research Funding.

Antibiotic resistance among enterococci and γ-proteobacteria is an increasing problem in healthcare settings. Dense colonization of the gut by antibiotic-resistant bacteria facilitates their spread between patients and also leads to bloodstream and other systemic infections. Antibiotic-mediated destruction of the intestinal microbiota and consequent loss of colonization resistance are critical factors leading to persistence and spread of antibiotic-resistant bacteria. The mechanisms underlying microbiota-mediated colonization resistance remain incompletely defined and are likely distinct for different antibiotic-resistant bacterial species. It is unclear whether enterococci or γ-proteobacteria, upon expanding to high density in the gut, confer colonization resistance against competing bacterial species. Herein, we demonstrate that dense intestinal colonization with vancomycin-resistant Enterococcus faecium (VRE) does not reduce in vivo growth of carbapenem-resistant Klebsiella pneumoniae. Reciprocally, K. pneumoniae does not impair intestinal colonization by VRE. In contrast, transplantation of a diverse fecal microbiota eliminates both VRE and K. pneumoniae from the gut. Fluorescence in situ hybridization demonstrates that VRE and K. pneumoniae localize to the same regions in the colon but differ with respect to stimulation and invasion of the colonic mucus layer. While VRE and K. pneumoniae occupy the same three-dimensional space within the gut lumen, their independent growth and persistence in the gut suggests that they reside in distinct niches that satisfy their specific in vivo metabolic needs., Author Summary Intestinal colonization precedes the development of disseminated infections and bacteremia by the nosocomial pathogens vancomycin-resistant Enterococcus (VRE) and carbapenem-resistant Klebsiella pneumoniae. Although antibiotic treatment renders mice susceptible to dense colonization by VRE or K. pneumoniae, it is unclear whether these microbes compete for space and resources in the gut. Our quantitative studies demonstrate that the density of intestinal colonization by either VRE or K. pneumoniae is unaffected by the presence of the other species, suggesting that they occupy separate niches. Using fluorescence in situ hybridization, we show that both bacterial species indeed occupy distinct niches but inhabit the same regions within the intestine. We find that K. pneumoniae, but not VRE, induces mucus production and invades the mucus layer adjacent to colonic epithelial cells, potentially leading to increased K. pneumoniae translocation to mesenteric lymph nodes. Despite their high colonization levels, both VRE and K. pneumoniae can be displaced from the intestinal lumen following transplantation of a healthy microbiota. Our study provides insight into the interactions between VRE and K. pneumoniae with each other and with their host.

Infection with the opportunistic enteric pathogen Clostridium difficile is an increasingly common clinical complication that follows antibiotic treatment-induced gut microbiota perturbation. Innate lymphoid cells (ILCs) are early responders to enteric pathogens; however, their role during C. difficile infection is undefined. To identify immune pathways that mediate recovery from C. difficile infection, we challenged C57BL/6, Rag1(-/-) (which lack T and B cells), and Rag2(-/-)Il2rg(-/-) (Ragγc(-/-)) mice (which additionally lack ILCs) with C. difficile. In contrast to Rag1(-/-) mice, ILC-deficient Ragγc(-/-) mice rapidly succumbed to infection. Rag1(-/-) but not Ragγc(-/-) mice upregulate expression of ILC1- or ILC3-associated proteins following C. difficile infection. Protection against infection was restored by transferring ILCs into Ragγc(-/-) mice. While ILC3s made a minor contribution to resistance, loss of IFN-γ or T-bet-expressing ILC1s in Rag1(-/-) mice increased susceptibility to C. difficile. These data demonstrate a critical role for ILC1s in defense against C. difficile.

Antibiotic administration disrupts the intestinal microbiota, increasing susceptibility to pathogens such as Clostridium difficile. Metronidazole or oral vancomycin can cure C. difficile infection, and administration of these agents to prevent C. difficile infection in high-risk patients, although not sanctioned by Infectious Disease Society of America guidelines, has been considered. The relative impacts of metronidazole and vancomycin on the intestinal microbiota and colonization resistance are unknown. We investigated the effect of brief treatment with metronidazole and/or oral vancomycin on susceptibility to C. difficile, vancomycin-resistant Enterococcus, carbapenem-resistant Klebsiella pneumoniae, and Escherichia coli infection in mice. Although metronidazole resulted in transient loss of colonization resistance, oral vancomycin markedly disrupted the microbiota, leading to prolonged loss of colonization resistance to C. difficile infection and dense colonization by vancomycin-resistant Enterococcus, K. pneumoniae, and E. coli. Our results demonstrate that vancomycin, and to a lesser extent metronidazole, are associated with marked intestinal microbiota destruction and greater risk of colonization by nosocomial pathogens.

Background The gut microbiome plays a role in the regulation of the immune system. Methods We prospectively enrolled 26 kidney transplant recipients and collected serial fecal specimens (N=85) during the first three months of transplantation. We characterized bacterial composition by polymerase chain reaction amplification of the 16S rRNA V4-V5 variable region and deep sequencing using the Illumina MiSeq platform. Results An increase in the relative abundance of Proteobacteria was observed in the posttransplantation specimens compared to pretransplantation specimens (P=0.04, Wilcoxon signed-rank test). In patients with posttransplant diarrhea, the mean(±standard deviation [SD]) Shannon diversity index was lower in those with diarrhea (N=6) than those without diarrhea (N=9) (2.5±0.3 vs. 3.4±0.8; P = 0.02, Wilcoxon rank-sum test). Principal coordinate analysis showed clear separation between the two groups, and linear discriminant analysis effect size (LEfSe) method revealed that Bacteroides, Ruminococcus, Coprococcus, and Dorea were significantly lower in the patients with diarrhea. Principal coordinate analysis also showed clear separation between the acute rejection (AR) group (N=3) and the no AR group (N=23) and the LEfSe method revealed significant differences between the two groups. Fecal abundance of Enterococcus was associated with Enterococcus urinary tract infection (UTI). The median Enterococcus fecal abundance was 24% (range, 8%-95%) in the three patients with Enterococcus UTI compared to 0% in the 23 patients without Enterococcus UTI (interquartile range, 0.00%-0.08%) (P=0.005, Wilcoxon rank-sum test). Conclusion Our pilot study identified significant alterations in the gut microbiota after kidney transplantation. Moreover, distinct microbiota structures were observed in allograft recipients with posttransplant diarrhea, AR, and Enterococcus UTI.

Highly diverse bacterial populations inhabit the gastrointestinal tract and modulate host inflammation and promote immune tolerance. In allogeneic hematopoietic stem cell transplantation (allo-HSCT), the gastrointestinal mucosa is damaged, and colonizing bacteria are impacted, leading to an impaired intestinal microbiota with reduced diversity. We examined the impact of intestinal diversity on subsequent mortality outcomes following transplantation. Fecal specimens were collected from 80 recipients of allo-HSCT at the time of stem cell engraftment. Bacterial 16S rRNA gene sequences were characterized, and microbial diversity was estimated using the inverse Simpson index. Subjects were classified into high, intermediate, and low diversity groups and assessed for differences in outcomes. Mortality outcomes were significantly worse in patients with lower intestinal diversity; overall survival at 3 years was 36%, 60%, and 67% for low, intermediate, and high diversity groups, respectively (P = .019, log-rank test). Low diversity showed a strong effect on mortality after multivariate adjustment for other clinical predictors (transplant related mortality: adjusted hazard ratio, 5.25; P = .014). In conclusion, the diversity of the intestinal microbiota at engraftment is an independent predictor of mortality in allo-HSCT recipients. These results indicate that the intestinal microbiota may be an important factor in the success or failure in allo-HSCT.

Bacteria causing infections in hospitalized patients are increasingly antibiotic resistant. Classical infection control practices are only partially effective at preventing spread of antibiotic-resistant bacteria within hospitals. Because the density of intestinal colonization by the highly antibiotic-resistant bacterium vancomycin-resistant Enterococcus (VRE) can exceed 10 9 organisms per gram of feces, even optimally implemented hygiene protocols often fail. Decreasing the density of intestinal colonization, therefore, represents an important approach to limit VRE transmission. We demonstrate that reintroduction of a diverse intestinal microbiota to densely VRE-colonized mice eliminates VRE from the intestinal tract. While oxygen-tolerant members of the microbiota are ineffective at eliminating VRE, administration of obligate anaerobic commensal bacteria to mice results in a billionfold reduction in the density of intestinal VRE colonization. 16S rRNA gene sequence analysis of intestinal bacterial populations isolated from mice that cleared VRE following microbiota reconstitution revealed that recolonization with a microbiota that contains Barnesiella correlates with VRE elimination. Characterization of the fecal microbiota of patients undergoing allogeneic hematopoietic stem cell transplantation demonstrated that intestinal colonization with Barnesiella confers resistance to intestinal domination and bloodstream infection with VRE. Our studies indicate that obligate anaerobic bacteria belonging to the Barnesiella genus enable clearance of intestinal VRE colonization and may provide novel approaches to prevent the spread of highly antibiotic-resistant bacteria.

Listeria monocytogenes is an important cause of foodborne infections, particularly in patients with hematologic malignancies, causing septicemia and meningoencephalitis. Although most strains of L. monocytogenes are antibiotic sensitive, antibiotic treatment is frustratingly ineffective at curing highly immunocompromised patients, resulting in a mortality rate of approximately 30%. The gut microbiota, which consists of commensal bacterial populations that inhabit the intestine, normally confers protection against orally acquired pathogens by exerting colonization resistance. Changes in microbiota composition induced by disease, pharmacological or antibiotic therapies can lead to dysbiosis, thereby impairing commensal-mediated colonization resistance and predisposing to infection. Our preliminary data show that bacterial species represented in a healthy microbiota can efficiently eliminate Listeria monocytogenes from the gut lumen, thus preventing the pathogen from translocating across the intestinal epithelium and spreading systemically. This mechanism of protection is particularly important in the context of congenic or acquired (chemotherapy-driven) immune deficiencies. Indeed, mice lacking T and B lymphocyets as well as innate lymphoid cells (Rag2/Il2rg Double Knockout mice, or Raggc), are highly susceptible to oral doses of Listeria that are non-lethal in WT mice. Although generally implicated in susceptibility to Listeriosis, the adaptive immune system is not involved in the in vivo clearance of the pathogen from the intestine, and the increased susceptibility to infection of these mice mainly results from the absence of innate immune cells producing interferon gamma at early stages, which leads to bacterial dissemination. Interestingly, Raggc mice are capable of surviving Listeria infection if challenged with low doses, which likely to mimicks the bacterial dosage acquired by immunocompromised patients. However, Raggc mice pre-treated with antibiotics experience uncontrolled intestinal growth of Listeria and subsequent systemic spread thus succumbing to low inoculum infection. This phenotype could be reproduced in mice treated with a combination of doxorubicin and cyclophosphamide, a cocktail often used in the treatment of cancer; although chemotherapy seemed to have an effect on the ability of the microbiota to restrict instestinal growth of Listeria, chemotherapy-treated mice became highly susceptible to low doses of Listeria only when pre-treated with antibiotics. Because antibiotics are often required for patients receiving cancer treatment, our these findings have potential clinical relevance. We are identifying specific commensal bacterial taxa and molecular mechanisms that mediate protection against L. monocytogenes. Our data indicate that Lactobacillus in the small intestine and Clostridium in the large intestine restrict Listeria growth in the gut lumen, thus lowering the bacterial burden and systemic spread. Germ-free (GF) mice reconstituted with a consortium of putative protective bacteria have greater resistance to infection than untreated GF mice or GF mice reconstituted with microbiota of antibiotic-treated mice, suggesting that gut commensals are independent and crucial contributors to host defense against Listeria. Our ultimate goal is to identify bacterial species that can be developed as probiotics for cancer patients to prevent intestinal L. monocytogenes carriage and infection. Combined shotgun sequencing and ex vivo assays on stool samples from cancer patients will be used to correlate loss of specific commensal bacteria with a predisposition to L. monocytogenes infection. Ultimately, this work will 1.) provide a novel approach to identifying subjects at high risk for L. monocytogenes infection and 2.) identify and characterize probiotic bacterial species to prevent and/or treat L. monocytogenes infections in cancer patients. Citation Format: Simone Becattini, Sohn G. Kim, Rebecca A. Carter, Lilan Ling, Ingrid M. Leiner, Eric G. Pamer. Identification of commensal bacterial strains that provide resistance to L. monocytogenes infection [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A082.

Introduction: Celecoxib is a chemopreventive agent that reduces adenomatous polyp number in the gastrointestinal tract of humans and mice and is thought to exert its effects exclusively through inhibition of COX-2. Bacteria have been shown to impact upon intestinal tumorigenesis in part by secretion of key metabolites. However, it is unknown whether celecoxib may be reducing polyp burden at least in part, through effects on the luminal microbiota or metabolome. This study tested whether celecoxib can alter the luminal microbiota and metabolome in association with reducing intestinal polyps in APCMin/+ mice, potentially providing a novel mechanism by which it exerts its chemopreventive effects. Methods: APCMin/+ and wild-type (WT) mice were co-housed for 2 weeks to normalize their microbiota, then individually housed and administered control or celecoxib-containing diet from 6 to 16 weeks of age. Feces were collected at 6, 11 and 16 weeks of age and ileal content and intestinal tissue (to quantify polyp burden) were collected at 16 weeks. Bacterial composition analysis on ileal content and feces was carried out using 16S rDNA sequencing and relative fecal metabolite levels were measured using UPLC/MS/MS and GC/MS. Results: As expected, administration of celecoxib for 10 weeks markedly reduced intestinal polyp burden in APCMin/+ mice. Concomitant with this effect, drug administration altered the luminal bacterial populations, a shift that also occurred in WT mice. These changes included increased Coriobacteriaceae and decreased Lactobacillaceae and Bifidobacteriaceae. Subsequent metabolomic analysis demonstrated that celecoxib caused a strong reduction in many fecal metabolites including glucose, amino acids, nucleotides and lipids. Ingenuity Pathway Analysis revealed that this change in metabolomic profile was associated with pathway perturbations that could contribute to reduced cell proliferation. Lastly, we showed that the observed changes were associated with a reduction in stem cell proliferation in normal intestinal crypts of APCMin/+ mice. Conclusions: These results demonstrate that celecoxib treatment profoundly altered the luminal microbiota and metabolome of mice and we hypothesize that these actions contribute to its chemopreventive activity. Further studies are warranted to confirm this hypothesis and assess whether our findings extend to humans and to other nonsteroidal anti-inflammatory drugs. Citation Format: David C. Montrose, Xi K. Zhou, Erin M. McNally, Erika Sue, Steven S. Gross, Nitai D. Leve, Edward D. Karoly, Lilan Ling, Eric G. Pamer, Andrew J. Dannenberg. Celecoxib alters the intestinal microbiota and metabolome in association with reducing polyp burden. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-302.

Commensal gut bacteria impact the host immune system and can influence disease processes in several organs, including the brain. However, it remains unclear whether the microbiota has an impact on the outcome of acute brain injury. Here we show that antibiotic-induced alterations in the intestinal flora reduce ischemic brain injury in mice, an effect transmissible by fecal transplants. Intestinal dysbiosis alters immune homeostasis in the small intestine, leading to an increase in regulatory T cells and a reduction in interleukin (IL)-17-positive γδ T cells through altered dendritic cell activity. Dysbiosis suppresses trafficking of effector T cells from the gut to the leptomeninges after stroke. Additionally, IL-10 and IL-17 are required for the neuroprotection afforded by intestinal dysbiosis. The findings reveal a previously unrecognized gut-brain axis and an impact of the intestinal flora and meningeal IL-17(+) γδ T cells on ischemic injury.

Clostridium difficile, an opportunistic pathogen that infects the gastrointestinal tract following perturbation of the microbiota, damages the intestinal epithelium and can cause debilitating and potentially fatal colitis. Antibiotic treatment of C. difficile is often ineffective and whether innate immune defenses can be exploited to promote C. difficile clearance is unknown. Innate lymphoid cells (ILCs) are early responders to enteric infection, however, their role during C. difficile infection is undefined. To identify immune pathways that mediate recovery from C. difficile infection, we compared C57BL/6, Rag1-/-, and Rag2-/- Il2rg-/- (Ragγc-/-) mice challenged with C. difficile. In contrast to C57BL/6 and Rag1-/- mice, ILC-deficient Ragγc-/- mice rapidly succumbed to infection. Protection against infection was restored by transferring ILCs into Ragγc-/- mice. While ILC3s made a minor contribution to resistance, loss of T-bet-expressing ILC1s or IFN-γ in Rag1-/- mice increased susceptibility to C. difficile. These data demonstrate a critical role for ILC1s in defense against C. difficile. Citation Format: Michael C. Abt, Brittany B. Lewis, Lilan Ling, Rebecca A. Carter, Boj Susac, Eric G. Pamer. Cooperative defense against acute Clostridium difficile infection is mediated by two innate lymphoid cell subsets. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A100.

The major causes of mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT) are relapse, graft-versus-host disease (GVHD), and infection. We have previously reported that changes in the intestinal flora can affect GVHD, bacteremia, and overall survival. As intestinal bacteria are potent modulators of systemic immune responses, and since GVHD is correlated with graft-versus-tumor activity, we hypothesized that components of the intestinal flora could be associated with relapse after allo-HSCT. We applied a biomarker-discovery approach and performed a retrospective observational analysis of 160 adults who received an unmodified (T-cell-replete) allograft. Patients were prospectively enrolled in a fecal biospecimen-collection protocol. For this analysis, we selected patients who had at least one specimen during the first 3 weeks following allo-HSCT. The primary diseases in this cohort were AML (37%), Non-Hodgkin's Lymphoma (33%), ALL (8%), MDS (7%), CLL (6%), Hodgkin's Lymphoma (6%), CML (2%), and myeloproliferative neoplasm (2%). The mean age of the patients was 52 years (range 21-75). They were conditioned with ablative (17%), reduced-intensity (64%), and nonmyeloablative (19%) regimens. They received grafts from cord blood (46%), unrelated adults (33%), or related adults (22%). Among adult grafts, 92% were from peripheral blood and 8% were from bone marrow. A census of the bacterial species in each stool sample was generated by 16S rRNA deep-sequencing as previously described (Jenq et al., BiolBone Marrow Transplant 2015). The area under the curve of bacterial abundance over time was used as a measure of each patient's cumulative exposure to each bacterial taxon. Bacterial taxa of each patient present at a frequency >1% were evaluated for association with the outcome of relapse or progression of disease within the first year after allo-HSCT using linear discriminant analysis of effect size (LEfSe), a common approach in microbiota studies (Segata et al., Genome Biology, 2011). Among the taxons most significantly associated with freedom from relapse were members of the human oral flora including Streptococcus anginosus. After stratifying the patients by median abundance, we found that those with higher abundance of this bacterium had less relapse after transplantation (Left figure, p = 0.0014). We also identified bacteria associated with increased risk of relapse, such as Enterococcus faecium (Right figure, p = 0.0103). We evaluated these bacteria as biomarkers in multivariate Cox models adjusted for three factors that were associated with relapse in this cohort: Refined Disease Risk Index (RDRI, Armand et al., Blood 2014), conditioning intensity, and graft source (cord blood vs. adult donor). Streptococcus anginosus predicted relapse in a multivariate model adjusted for all three factors (HR 0.39, 95% CI 0.16-0.96, p = 0.041). Enterococcus faecium predicted relapse in a model adjusted for RDRI and conditioning intensity but failed to do so in a model additionally adjusted for graft source. In this analysis there was no formal adjustment for multiple comparisons; these data are now being validated in an additional cohort of patients whose samples are being sequenced. Finally, although we have previously reported that low bacterial diversity is associated with decreased overall survival after allo-HSCT (Taur et al., Blood 2014), we did not find an association between bacterial diversity and relapse as assessed by reciprocal Simpson diversity index (p > 0.1). Thus, the results of this retrospective analysis have identified an association between relapse after allo-HSCT and the abundance of two bacteria in the intestinal flora. These might serve as potential novel diagnostics or therapeutic targets to prevent relapse and improve overall survival after allo-HSCT. Figure 1. Figure 1. Disclosures Peled: Merck: Research Funding. Giralt:SANOFI: Consultancy, Honoraria, Research Funding; TAKEDA: Consultancy, Honoraria, Research Funding; AMGEN: Consultancy, Research Funding; JAZZ: Consultancy, Honoraria, Research Funding, Speakers Bureau; CELGENE: Consultancy, Honoraria, Research Funding. Perales:Merck: Honoraria; Takeda: Honoraria; Amgen: Honoraria; Astellas: Honoraria; NMDP: Membership on an entity's Board of Directors or advisory committees. van den Brink:Boehringer Ingelheim: Consultancy, Other: Advisory board attendee; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tobira Therapeutics: Other: Advisory board attendee; Regeneron: Honoraria; Merck: Honoraria.