Exploring Rifamycin Inactivation from the Soil Microbiome

Our battle against pathogens has become a challenge due to the rise in antibiotic resistance and the dwindling number of new antibiotics entering the clinic. Most antibiotics owe their origins to soil bacteria, which have been producing these natural products for millennia. The rifamycins are products of actinomycetes and semisynthetic derivatives of these have been very successful in the clinic. Rifampin (RIF) has been a cornerstone agent against tuberculosis for over 50 years. In the clinic, pathogens typically develop RIF resistance by mutation of the drug. Nonetheless, a number of diverse RIF resistance mechanisms have been described, including enzymatic inactivation. Environmental bacteria are multidrug resistant, likely due to sharing the same niche as antibiotic producers and represent a reservoir of ancient resistance determinants. Furthermore, these resistance determinants have been linked to pathogens. Exploring the antibiotic resistome, the collection of all antibiotic resistance determinants from the global microbiota, reveals the diversity and evolution of resistance and provides insight on vulnerabilities of our current antibiotics. Herein, I describe a diverse collection of RIF-inactivating mechanisms from soil actinomycetes. I identified heretofore unknown RIF glycosyltransferase and RIF phosphotransferase genes (rgt and rph, respectively). RGT and RPH enzymes display broad rifamycin specificity and contribute to high-level resistance. Interestingly, RIF-sensitive Gram-positive pathogens are carriers of RPH, highlighting the existence of a ‘silent’ resistome in clinically relevant bacteria and emphasize the importance of studying resistance from environmental bacteria. Furthermore, I identified a conserved upstream DNA motif associated with RIF-inactivating genes from actinomycetes and demonstrate its role in RIF-responsive gene regulation. Finally, I explore the use of a RIF-resistance guided approach to identify novel rifamycin producing bacteria. This study expands the rifamycin resistome, provides evidence of vulnerabilities of our current arsenal of rifamycin antibiotics, and offers a strategy to identify new members of this family natural product family.ThesisDoctor of Science (PhD

Expansion of a bacterial operon during cancer treatment ameliorates fluoropyrimidine toxicity

Dose-limiting toxicities remain a major barrier to drug development and therapy, revealing the limited predictive power of human genetics. Here, we demonstrate the utility of a more comprehensive approach to studying drug toxicity through longitudinal profiling of the human gut microbiome during colorectal cancer (CRC) treatment (NCT04054908) coupled to cell culture and mouse experiments. Substantial shifts in gut microbial community structure during oral fluoropyrimidine treatment across multiple patient cohorts, in mouse small and large intestinal contents, and in patient-derived ex vivo communities were revealed by 16S rRNA gene sequencing. Metagenomic sequencing revealed marked shifts in pyrimidine-related gene abundance during oral fluoropyrimidine treatment, including enrichment of the preTA operon, which was sufficient for the inactivation of active metabolite 5-fluorouracil (5-FU). preTA+ bacteria depleted 5-FU in gut microbiota grown ex vivo and in the mouse distal gut. Germ-free and antibiotic-treated mice experienced increased fluoropyrimidine toxicity, which was rescued by colonization with the mouse gut microbiota, preTA+ Escherichia coli, or preTA-high stool from patients with CRC. Last, preTA abundance was negatively associated with fluoropyrimidine toxicity in patients. Together, these data support a causal, clinically relevant interaction between a human gut bacterial operon and the dose-limiting side effects of cancer treatment. Our approach may be generalizable to other drugs, including cancer immunotherapies, and provides valuable insights into host-microbiome interactions in the context of disease

Mirror, mirror on the wall: which microbiomes will help heal them all?

BACKGROUND: Clinicians have known for centuries that there is substantial variability between patients in their response to medications—some individuals exhibit a miraculous recovery while others fail to respond at all. Still others experience dangerous side effects. The hunt for the factors responsible for this variation has been aided by the ability to sequence the human genome, but this just provides part of the picture. Here, we discuss the emerging field of study focused on the human microbiome and how it may help to better predict drug response and improve the treatment of human disease. DISCUSSION: Various clinical disciplines characterize drug response using either continuous or categorical descriptors that are then correlated to environmental and genetic risk factors. However, these approaches typically ignore the microbiome, which can directly metabolize drugs into downstream metabolites with altered activity, clearance, and/or toxicity. Variations in the ability of each individual’s microbiome to metabolize drugs may be an underappreciated source of differences in clinical response. Complementary studies in humans and animal models are necessary to elucidate the mechanisms responsible and to test the feasibility of identifying microbiome-based biomarkers of treatment outcomes. SUMMARY: We propose that the predictive power of genetic testing could be improved by taking a more comprehensive view of human genetics that encompasses our human and microbial genomes. Furthermore, unlike the human genome, the microbiome is rapidly altered by diet, pharmaceuticals, and other interventions, providing the potential to improve patient care by re-shaping our associated microbial communities

A rifamycin inactivating phosphotransferase family shared by environmental and pathogenic bacteria

Significance Environmental microorganisms are a source of diverse antibiotic resistance determinants. With the appropriate selection pressure, these resistance genes can be mobilized to clinically relevant pathogens. Identifying and characterizing elements of the environmental antibiotic resistome provides an early warning of what we may expect to encounter in the clinic. We uncover a conserved genetic element associated with various rifamycin antibiotic-inactivating mechanisms. This element led to the identification of a new resistance gene and associated enzyme responsible for inactivating rifamycin antibiotics by phosphorylation. Cryptic orthologous genes are also found in pathogenic bacteria but remain susceptible to the drug. This study reveals a new antibiotic resistance protein family and the unexpected prevalence of a silent rifamycin resistome among pathogenic bacteria.</jats:p

The global anaerobic metabolism regulator fnr is necessary for the degradation of food dyes and drugs by Escherichia coli.

The microbiome is an underappreciated contributor to intestinal drug metabolism with broad implications for drug efficacy and toxicity. While considerable progress has been made toward identifying the gut bacterial genes and enzymes involved, the role of environmental factors in shaping their activity remains poorly understood. Here, we focus on the gut bacterial reduction of azo bonds (R-N = N-R), found in diverse chemicals in both food and drugs. Surprisingly, the canonical azoR gene in Escherichia coli was dispensable for azo bond reduction. Instead, azoreductase activity was controlled by the fumarate and nitrate reduction (fnr) regulator, consistent with a requirement for the anoxic conditions found within the gastrointestinal tract. Paired transcriptomic and proteomic analysis of the fnr regulon revealed that in addition to altering the expression of multiple reductases, FNR is necessary for the metabolism of L-Cysteine to hydrogen sulfide, enabling the degradation of azo bonds. Furthermore, we found that FNR indirectly regulates this process through the small noncoding regulatory RNA fnrS. Taken together, these results show how gut bacteria sense and respond to their intestinal environment to enable the metabolism of chemical groups found in both dietary and pharmaceutical compounds. IMPORTANCE This work has broad relevance due to the ubiquity of dyes containing azo bonds in food and drugs. We report that azo dyes can be degraded by human gut bacteria through both enzymatic and nonenzymatic mechanisms, even from a single gut bacterial species. Furthermore, we revealed that environmental factors, oxygen, and L-Cysteine control the ability of E. coli to degrade azo dyes due to their impacts on bacterial transcription and metabolism. These results open up new opportunities to manipulate the azoreductase activity of the gut microbiome through the manipulation of host diet, suggest that azoreductase potential may be altered in patients suffering from gastrointestinal disease, and highlight the importance of studying bacterial enzymes for drug metabolism in their natural cellular and ecological context

How to Determine the Role of the Microbiome in Drug Disposition.

With a paradigm shift occurring in health care toward personalized and precision medicine, understanding the numerous environmental factors that impact drug disposition is of paramount importance. The highly diverse and variant nature of the human microbiome is now recognized as a factor driving interindividual variation in therapeutic outcomes. The purpose of this review is to provide a practical guide on methodology that can be applied to study the effects of microbes on the absorption, distribution, metabolism, and excretion of drugs. We also highlight recent examples of how these methods have been successfully applied to help build the basis for researching the intersection of the microbiome and pharmacology. Although in vitro and in vivo preclinical models are highlighted, these methods are also relevant in late-phase drug development or even as a part of routine after-market surveillance. These approaches will aid in filling major knowledge gaps for both current and upcoming therapeutics with the long-term goal of achieving a new type of knowledge-based medicine that integrates data on the host and the microbiome