Nora C. Toussaint

Precision microbiome reconstitution restores bile acid mediated resistance to Clostridium difficile

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.

Intestinal Microbiota Containing Barnesiella Species Cures Vancomycin-Resistant Enterococcus faecium Colonization

ABSTRACT 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 109 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.

Ecological modeling from time-series inference: insight into dynamics and stability of intestinal microbiota.

The intestinal microbiota is a microbial ecosystem of crucial importance to human health. Understanding how the microbiota confers resistance against enteric pathogens and how antibiotics disrupt that resistance is key to the prevention and cure of intestinal infections. We present a novel method to infer microbial community ecology directly from time-resolved metagenomics. This method extends generalized Lotka–Volterra dynamics to account for external perturbations. Data from recent experiments on antibiotic-mediated Clostridium difficile infection is analyzed to quantify microbial interactions, commensal-pathogen interactions, and the effect of the antibiotic on the community. Stability analysis reveals that the microbiota is intrinsically stable, explaining how antibiotic perturbations and C. difficile inoculation can produce catastrophic shifts that persist even after removal of the perturbations. Importantly, the analysis suggests a subnetwork of bacterial groups implicated in protection against C. difficile. Due to its generality, our method can be applied to any high-resolution ecological time-series data to infer community structure and response to external stimuli.

Proton Pump Inhibitors Alter Specific Taxa in the Human Gastrointestinal Microbiome: A Crossover Trial.

We conducted an open-label crossover trial to test whether proton pump inhibitors (PPIs) affect the gastrointestinal microbiome to facilitate Clostridium difficile infection (CDI). Twelve healthy volunteers each donated 2 baseline fecal samples, 4 weeks apart (at weeks 0 and 4). They then took PPIs for 4 weeks (40 mg omeprazole, twice daily) and fecal samples were collected at week 8. Six individuals took the PPIs for an additional 4 weeks (from week 8 to 12) and fecal samples were collected from all subjects at week 12. Samples were analyzed by 16S ribosomal RNA gene sequencing. We found no significant within-individual difference in microbiome diversity when we compared changes during baseline vs changes on PPIs. There were, however, significant changes during PPI use in taxa associated with CDI (increased Enterococcaceae and Streptococcaceae, decreased Clostridiales) and taxa associated with gastrointestinal bacterial overgrowth (increased Micrococcaceae and Staphylococcaceae). In a functional analysis, there were no changes in bile acids on PPIs, but there was an increase in genes involved in bacterial invasion. These alterations could provide a mechanism by which PPIs predispose to CDI. ClinicalTrials.gov ID NCT01901276.

Loss of Microbiota-Mediated Colonization Resistance to Clostridium difficile Infection With Oral Vancomycin Compared With Metronidazole

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.

Gut microbial community structure and complications after kidney transplantation: a pilot study.

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.

Gut Microbiota and Tacrolimus Dosing in Kidney Transplantation

Tacrolimus dosing to establish therapeutic levels in recipients of organ transplants is a challenging task because of much interpatient and intrapatient variability in drug absorption, metabolism, and disposition. In view of the reported impact of gut microbial species on drug metabolism, we investigated the relationship between the gut microbiota and tacrolimus dosing requirements in this pilot study of adult kidney transplant recipients. Serial fecal specimens were collected during the first month of transplantation from 19 kidney transplant recipients who either required a 50% increase from initial tacrolimus dosing during the first month of transplantation (Dose Escalation Group, n=5) or did not require such an increase (Dose Stable Group, n=14). We characterized bacterial composition in the fecal specimens by deep sequencing of the PCR amplified 16S rRNA V4-V5 region and we investigated the hypothesis that gut microbial composition is associated with tacrolimus dosing requirements. Initial tacrolimus dosing was similar in the Dose Escalation Group and in the Stable Group (4.2±1.1 mg/day vs. 3.8±0.8 mg/day, respectively, P=0.61, two-way between-group ANOVA using contrasts) but became higher in the Dose Escalation Group than in the Dose Stable Group by the end of the first transplantation month (9.6±2.4 mg/day vs. 3.3±1.5 mg/day, respectively, P<0.001). Our systematic characterization of the gut microbial composition identified that fecal Faecalibacterium prausnitzii abundance in the first week of transplantation was 11.8% in the Dose Escalation Group and 0.8% in the Dose Stable Group (P=0.002, Wilcoxon Rank Sum test, P<0.05 after Benjamini-Hochberg correction for multiple hypotheses). Fecal Faecalibacterium prausnitzii abundance in the first week of transplantation was positively correlated with future tacrolimus dosing at 1 month (R=0.57, P=0.01) and had a coefficient±standard error of 1.0±0.6 (P=0.08) after multivariable linear regression. Our novel observations may help further explain inter-individual differences in tacrolimus dosing to achieve therapeutic levels.

Combined mutation in Vhl, Trp53 and Rb1 causes clear cell renal cell carcinoma in mice

Clear cell renal cell carcinomas (ccRCCs) frequently exhibit inactivation of the von Hippel–Lindau tumor-suppressor gene, VHL, and often harbor multiple copy-number alterations in genes that regulate cell cycle progression. We show here that modeling these genetic alterations by combined deletion of Vhl, Trp53 and Rb1 specifically in renal epithelial cells in mice caused ccRCC. These tumors arose from proximal tubule epithelial cells and shared molecular markers and mRNA expression profiles with human ccRCC. Exome sequencing revealed that mouse and human ccRCCs exhibit recurrent mutations in genes associated with the primary cilium, uncovering a mutational convergence on this organelle and implicating a subset of ccRCCs as genetic ciliopathies. Different mouse tumors responded differently to standard therapies for advanced human ccRCC, mimicking the range of clinical behaviors in the human disease. Inhibition of hypoxia-inducible factor (HIF)-α transcription factors with acriflavine as third-line therapy had therapeutic effects in some tumors, providing preclinical evidence for further investigation of HIF-α inhibition as a ccRCC treatment. This autochthonous mouse ccRCC model represents a tool to investigate the biology of ccRCC and to identify new treatment strategies.

Bioinformatics for precision oncology

Abstract Molecular profiling of tumor biopsies plays an increasingly important role not only in cancer research, but also in the clinical management of cancer patients. Multi-omics approaches hold the promise of improving diagnostics, prognostics and personalized treatment. To deliver on this promise of precision oncology, appropriate bioinformatics methods for managing, integrating and analyzing large and complex data are necessary. Here, we discuss the specific requirements of bioinformatics methods and software that arise in the setting of clinical oncology, owing to a stricter regulatory environment and the need for rapid, highly reproducible and robust procedures. We describe the workflow of a molecular tumor board and the specific bioinformatics support that it requires, from the primary analysis of raw molecular profiling data to the automatic generation of a clinical report and its delivery to decision-making clinical oncologists. Such workflows have to various degrees been implemented in many clinical trials, as well as in molecular tumor boards at specialized cancer centers and university hospitals worldwide. We review these and more recent efforts to include other high-dimensional multi-omics patient profiles into the tumor board, as well as the state of clinical decision support software to translate molecular findings into treatment recommendations.

Genomic and Geographic Context for the Evolution of High-Risk Carbapenem-Resistant Enterobacter cloacae Complex Clones ST171 and ST78

ABSTRACT Recent reports have established the escalating threat of carbapenem-resistant Enterobacter cloacae complex (CREC). Here, we demonstrate that CREC has evolved as a highly antibiotic-resistant rather than highly virulent nosocomial pathogen. Applying genomics and Bayesian phylogenetic analyses to a 7-year collection of CREC isolates from a northern Manhattan hospital system and to a large set of publicly available, geographically diverse genomes, we demonstrate clonal spread of a single clone, ST171. We estimate that two major clades of epidemic ST171 diverged prior to 1962, subsequently spreading in parallel from the Northeastern to the Mid-Atlantic and Midwestern United States and demonstrating links to international sites. Acquisition of carbapenem and fluoroquinolone resistance determinants by both clades preceded widespread use of these drugs in the mid-1980s, suggesting that antibiotic pressure contributed substantially to its spread. Despite a unique mobile repertoire, ST171 isolates showed decreased virulence in vitro. While a second clone, ST78, substantially contributed to the emergence of CREC, it encompasses diverse carbapenemase-harboring plasmids, including a potentially hypertransmissible IncN plasmid, also present in other sequence types. Rather than heightened virulence, CREC demonstrates lineage-specific, multifactorial adaptations to nosocomial environments coupled with a unique potential to acquire and disseminate carbapenem resistance genes. These findings indicate a need for robust surveillance efforts that are attentive to the potential for local and international spread of high-risk CREC clones. IMPORTANCE Carbapenem-resistant Enterobacter cloacae complex (CREC) has emerged as a formidable nosocomial pathogen. While sporadic acquisition of plasmid-encoded carbapenemases has been implicated as a major driver of CREC, ST171 and ST78 clones demonstrate epidemic potential. However, a lack of reliable genomic references and rigorous statistical analyses has left many gaps in knowledge regarding the phylogenetic context and evolutionary pathways of successful CREC. Our reconstruction of recent ST171 and ST78 evolution represents a significant addition to current understanding of CREC and the directionality of its spread from the Eastern United States to the northern Midwestern United States with links to international collections. Our results indicate that the remarkable ability of E. cloacae to acquire and disseminate cross-class antibiotic resistance rather than virulence determinants, coupled with its ability to adapt under conditions of antibiotic pressure, likely led to the wide dissemination of CREC. IMPORTANCE Carbapenem-resistant Enterobacter cloacae complex (CREC) has emerged as a formidable nosocomial pathogen. While sporadic acquisition of plasmid-encoded carbapenemases has been implicated as a major driver of CREC, ST171 and ST78 clones demonstrate epidemic potential. However, a lack of reliable genomic references and rigorous statistical analyses has left many gaps in knowledge regarding the phylogenetic context and evolutionary pathways of successful CREC. Our reconstruction of recent ST171 and ST78 evolution represents a significant addition to current understanding of CREC and the directionality of its spread from the Eastern United States to the northern Midwestern United States with links to international collections. Our results indicate that the remarkable ability of E. cloacae to acquire and disseminate cross-class antibiotic resistance rather than virulence determinants, coupled with its ability to adapt under conditions of antibiotic pressure, likely led to the wide dissemination of CREC.