Mark B. Smith

Ecology drives a global network of gene exchange connecting the human microbiome

Horizontal gene transfer (HGT), the acquisition of genetic material from non-parental lineages, is known to be important in bacterial evolution. In particular, HGT provides rapid access to genetic innovations, allowing traits such as virulence, antibiotic resistance and xenobiotic metabolism to spread through the human microbiome. Recent anecdotal studies providing snapshots of active gene flow on the human body have highlighted the need to determine the frequency of such recent transfers and the forces that govern these events. Here we report the discovery and characterization of a vast, human-associated network of gene exchange, large enough to directly compare the principal forces shaping HGT. We show that this network of 10,770 unique, recently transferred (more than 99% nucleotide identity) genes found in 2,235 full bacterial genomes, is shaped principally by ecology rather than geography or phylogeny, with most gene exchange occurring between isolates from ecologically similar, but geographically separated, environments. For example, we observe 25-fold more HGT between human-associated bacteria than among ecologically diverse non-human isolates (P = 3.0 × 10−270). We show that within the human microbiome this ecological architecture continues across multiple spatial scales, functional classes and ecological niches with transfer further enriched among bacteria that inhabit the same body site, have the same oxygen tolerance or have the same ability to cause disease. This structure offers a window into the molecular traits that define ecological niches, insight that we use to uncover sources of antibiotic resistance and identify genes associated with the pathology of meningitis and other diseases.

Fecal Microbiota Transplant for Relapsing Clostridium difficile Infection Using a Frozen Inoculum From Unrelated Donors: A Randomized, Open-Label, Controlled Pilot Study

BACKGROUND Recurrent Clostridium difficile infection (CDI) with poor response to standard antimicrobial therapy is a growing medical concern. We aimed to investigate the outcomes of fecal microbiota transplant (FMT) for relapsing CDI using a frozen suspension from unrelated donors, comparing colonoscopic and nasogastric tube (NGT) administration. METHODS Healthy volunteer donors were screened and a frozen fecal suspension was generated. Patients with relapsing/refractory CDI were randomized to receive an infusion of donor stools by colonoscopy or NGT. The primary endpoint was clinical resolution of diarrhea without relapse after 8 weeks. The secondary endpoint was self-reported health score using standardized questionnaires. RESULTS A total of 20 patients were enrolled, 10 in each treatment arm. Patients had a median of 4 (range, 2-16) relapses prior to study enrollment, with 5 (range, 3-15) antibiotic treatment failures. Resolution of diarrhea was achieved in 14 patients (70%) after a single FMT (8 of 10 in the colonoscopy group and 6 of 10 in the NGT group). Five patients were retreated, with 4 obtaining cure, resulting in an overall cure rate of 90%. Daily number of bowel movements changed from a median of 7 (interquartile range [IQR], 5-10) the day prior to FMT to 2 (IQR, 1-2) after the infusion. Self-ranked health score improved significantly, from a median of 4 (IQR, 2-6) before transplant to 8 (IQR, 5-9) after transplant. No serious or unexpected adverse events occurred. CONCLUSIONS In our initial feasibility study, FMT using a frozen inoculum from unrelated donors is effective in treating relapsing CDI. NGT administration appears to be as effective as colonoscopic administration. CLINICAL TRIALS REGISTRATION NCT01704937.

Natural Bacterial Communities Serve as Quantitative Geochemical Biosensors

ABSTRACT Biological sensors can be engineered to measure a wide range of environmental conditions. Here we show that statistical analysis of DNA from natural microbial communities can be used to accurately identify environmental contaminants, including uranium and nitrate at a nuclear waste site. In addition to contamination, sequence data from the 16S rRNA gene alone can quantitatively predict a rich catalogue of 26 geochemical features collected from 93 wells with highly differing geochemistry characteristics. We extend this approach to identify sites contaminated with hydrocarbons from the Deepwater Horizon oil spill, finding that altered bacterial communities encode a memory of prior contamination, even after the contaminants themselves have been fully degraded. We show that the bacterial strains that are most useful for detecting oil and uranium are known to interact with these substrates, indicating that this statistical approach uncovers ecologically meaningful interactions consistent with previous experimental observations. Future efforts should focus on evaluating the geographical generalizability of these associations. Taken as a whole, these results indicate that ubiquitous, natural bacterial communities can be used as in situ environmental sensors that respond to and capture perturbations caused by human impacts. These in situ biosensors rely on environmental selection rather than directed engineering, and so this approach could be rapidly deployed and scaled as sequencing technology continues to become faster, simpler, and less expensive. IMPORTANCE Here we show that DNA from natural bacterial communities can be used as a quantitative biosensor to accurately distinguish unpolluted sites from those contaminated with uranium, nitrate, or oil. These results indicate that bacterial communities can be used as environmental sensors that respond to and capture perturbations caused by human impacts. Here we show that DNA from natural bacterial communities can be used as a quantitative biosensor to accurately distinguish unpolluted sites from those contaminated with uranium, nitrate, or oil. These results indicate that bacterial communities can be used as environmental sensors that respond to and capture perturbations caused by human impacts.

Transfer of noncoding DNA drives regulatory rewiring in bacteria

Significance The rapid pace of evolution in bacteria is widely attributed to the promiscuous horizontal transfer and recombination of protein-coding genes. However, it has not been investigated if the same forces also drive the evolution of noncoding regulatory regions. Here, we establish that regulatory regions can “switch” between nonhomologous alternatives and that switching is ubiquitous, occurring across the bacterial domain. We show that regulatory switching has a strong impact on promoter architecture and expression divergence. Further, we demonstrate that regulatory transfer facilitates rapid phenotypic diversification of a human pathogen. This regulatory mobility enables bacterial genes to access a vast pool of potential regulatory elements, facilitating efficient exploration of the regulatory landscape. Understanding the mechanisms that generate variation is a common pursuit unifying the life sciences. Bacteria represent an especially striking puzzle, because closely related strains possess radically different metabolic and ecological capabilities. Differences in protein repertoire arising from gene transfer are currently considered the primary mechanism underlying phenotypic plasticity in bacteria. Although bacterial coding plasticity has been extensively studied in previous decades, little is known about the role that regulatory plasticity plays in bacterial evolution. Here, we show that bacterial genes can rapidly shift between multiple regulatory modes by acquiring functionally divergent nonhomologous promoter regions. Through analysis of 270,000 regulatory regions across 247 genomes, we demonstrate that regulatory “switching” to nonhomologous alternatives is ubiquitous, occurring across the bacterial domain. Using comparative transcriptomics, we show that at least 16% of the expression divergence between Escherichia coli strains can be explained by this regulatory switching. Further, using an oligonucleotide regulatory library, we establish that switching affects bacterial promoter architecture. We provide evidence that regulatory switching can occur through horizontal regulatory transfer, which allows regulatory regions to move across strains, and even genera, independently from the genes they regulate. Finally, by experimentally characterizing the fitness effect of a regulatory transfer on a pathogenic E. coli strain, we demonstrate that regulatory switching elicits important phenotypic consequences. Taken together, our findings expose previously unappreciated regulatory plasticity in bacteria and provide a gateway for understanding bacterial phenotypic variation and adaptation.

Clostridium difficile associated risk of death score (CARDS): a novel severity score to predict mortality among hospitalised patients with C. difficile infection

Clostridium difficile infection (CDI) is a public health threat and associated with significant mortality. However, there is a paucity of objectively derived CDI severity scoring systems to predict mortality.

Computational Methods for High-Throughput Comparative Analyses of Natural Microbial Communities

One of the most widely employed methods in metagenomics is the amplification and sequencing of the highly conserved ribosomal RNA (rRNA) genes from organisms in complex microbial communities. rRNA surveys, typically using the 16S rRNA gene for prokaryotic identification, provide information about the total diversity and taxonomic affiliation of organisms present in a sample. Greatly enhanced by high-throughput sequencing, these surveys have uncovered the remarkable diversity of uncultured organisms and revealed unappreciated ecological roles ranging from nutrient cycling to human health. This chapter outlines the best practices for comparative analyses of microbial community surveys. We explain how to transform raw data into meaningful units for further analysis and discuss how to calculate sample diversity and community distance metrics. Finally, we outline how to find associations of species with specific metadata and true correlations between species from compositional data. We focus on data generated by next-generation sequencing platforms, using the Illumina platform as a test case, because of its widespread use especially among researchers just entering the field.

Strain Tracking Reveals the Determinants of Bacterial Engraftment in the Human Gut Following Fecal Microbiota Transplantation

Fecal microbiota transplantation (FMT) from healthy donor to patient is a treatment for microbiome-associated diseases. Although the success of FMT requires donor bacteria to engraft in the patients gut, the forces governing engraftment in humans are unknown. Here we use an ongoing clinical experiment, the treatment of recurrent Clostridium difficile infection, to uncover the rules of engraftment in humans. We built a statistical model that predicts which bacterial species will engraft in a given host, and developed Strain Finder, a method to infer strain genotypes and track them over time. We find that engraftment can be predicted largely from the abundance and phylogeny of bacteria in the donor and the pre-FMT patient. Furthermore, donor strains within a species engraft in an all-or-nothing manner and previously undetected strains frequently colonize patients receiving FMT. We validated these findings for metabolic syndrome, suggesting that the same principles of engraftment extend to other indications.

The regulation of fecal microbiota for transplantation: An international perspective for policy and public health

Abstract Clostridium difficile is the most common hospital-acquired pathogen in the US, and recurrent C. difficile infection (CDI) is a major public health issue. Twenty per cent of CDI patients experience recurrence, and their risk of recurrence rises with each failure to achieve clinical resolution. Fecal microbiota transplantation (FMT) is a remarkably efficacious treatment for recurrent CDI. However, national health agencies are grappling with the appropriate regulatory paradigm to apply to this innovative treatment. Current FMT regulations in the US, Canada, Western Europe, Australia, and China are in varying degrees of flux, although many regulators are choosing to apply the drug and biologic framework. FMT regulations should allow recurrent CDI patients safe access to this treatment as research continues. Regulating FMT like a drug or biologic, although most convenient from a legal perspective, overly restricts access while under-regulating the methods by which the stool is screened, processed, stored, and used. Human tissue and tissue-based products regulations could achieve the desired level and kind of oversight, but fecal microbiota for transplantation fail to meet applicable statutory definitions. A custom regulatory solution would be more appropriate, but many pathways that regulators may take to achieve this goal require time and resources for health agencies to develop.

Profiling Living Bacteria Informs Preparation of Fecal Microbiota Transplantations.

Fecal microbiota transplantation is a compelling treatment for recurrent Clostridium difficile infections, with potential applications against other diseases associated with changes in gut microbiota. But variability in fecal bacterial communities—believed to be the therapeutic agent—can complicate or undermine treatment efficacy. To understand the effects of transplant preparation methods on living fecal microbial communities, we applied a DNA-sequencing method (PMA-seq) that uses propidium monoazide (PMA) to differentiate between living and dead fecal microbes, and we created an analysis pipeline to identify individual bacteria that change in abundance between samples. We found that oxygen exposure degraded fecal bacterial communities, whereas freeze-thaw cycles and lag time between donor defecation and transplant preparation had much smaller effects. Notably, the abundance of Faecalibacterium prausnitzii—an anti-inflammatory commensal bacterium whose absence is linked to inflammatory bowel disease—decreased with oxygen exposure. Our results indicate that some current practices for preparing microbiota transplant material adversely affect living fecal microbial content and highlight PMA-seq as a valuable tool to inform best practices and evaluate the suitability of clinical fecal material.

Sa1064 The International Public Stool Bank: A Scalable Model for Standardized Screening and Processing of Donor Stool for Fecal Microbiota Transplantation

the inpatient quality indicators of length of stay (LOS), 30-day re-admission rates, and mortality for patients admitted to our academic medical center for gastrointestinal bleeding. Methods: We retrospectively evaluated admissions for gastrointestinal bleeding based on ICD-9-CM codes from May 2011-June 2013 at the University of Miami Hospital (Miami, FL). The patients were primarily managed by one of four types of inpatient service as follows: GI teaching service, academic medicine teaching service, private hospitalist, or surgical service. Patients initially admitted to an intensive care unit from the emergency department were excluded. To account for potential differences in severity of GI bleeding between services, patients were matched by propensity score based on their age, location of bleed (upper GI bleed vs lower GI bleed), and whether the bleed was secondary to portal hypertension. Results: 600 hospital admissions for GI bleeding were included (GI 84, academic 44, private 436, surgical 36). No difference was noted in the mean LOS among groups (GI 4.72 days, academic 5.56 days, private 5.96 days, and surgeons 6 days, ANOVA F=1.44, p= 0.23). No difference was observed in the 30 day re-admission rate (GI 1.85%, academic 5.13%, private 4.51%, surgeons 3.03%, Pearsons chi-squared 1.03, p=0.79). No difference was seen in the in-hospital mortality rate (GI 1.89%, academic 2.56%, private 2.13%, surgeons 0%, Pearsons chi-squared 0.77, p=0.86). Following propensity score matching, 491 patients were evaluated. No differences were noted in LOS (ANOVA, F=2.95, p= 0.053) or re-admission rates (LR Chi-squared 1.64, p=0.44) among groups. Conclusion: No significant difference was observed in inpatient quality indicators for a GI specialty service at an academic center. However, small numbers may have prevented these values from reaching significance as trends were observed favoring the GI team with LOS and readmission, making a larger sample size and a cost evaluation important next steps in this evaluation process.