A Mechanism of Antimicrobial Resistance and a Mitigation Strategy

ABSTRACT OF THE DISSERTATION A Mechanism of Antimicrobial Resistance and a Mitigation Strategy by Christopher Bulow Doctor of Philosophy in Biology and Biomedical Sciences Molecular Genetics and Genomics Washington University in St. Louis, 2018 Professor Gautam Dantas, Chair The ability to treat infections, perform surgery, and administer immunosuppressants and chemotherapy depends on effective antibiotics. The emergence and spread of antimicrobial resistance is far outpacing the development of new therapies1-3 threatening to thrust medicine into a post-antibiotic era4. Many mechanisms of antimicrobial action and of antimicrobial resistance remain poorly understood as drug development struggles to keep pace. As resistance develops, the human gut serves as a reservoir and provides ample opportunities for resistance gene transmission between commensal and pathogenic bacteria5-11. Once resistant organisms colonize the gut, they can persist for extended durations even without continued antimicrobial exposure6-12. New approaches are necessary to prevent or reverse colonization with resistant organisms. This work takes two important steps to addressing the antimicrobial resistance crisis: 1) Understanding an antimicrobial mechanism of action and a corresponding mechanism of resistance and 2) Developing an approach to prevent or reverse colonization of human hosts with resistant organisms. Daptomycin, a broad spectrum antibiotic used for treating multi-drug resistant Gram-positive infections, is experiencing clinical failure against important infectious agents including Corynebacterium striatum, an opportunistic pathogen and skin commensal. The recent transition of daptomycin to generic status is projected to dramatically increase availability, use, and clinical failure. Here we confirm the genetic mechanism of high-level daptomycin resistance (HLDR, MIC \u3e 256 g/mL) in C. striatum, which evolved within a patient during daptomycin therapy. This work demonstrates that loss of function mutation in pgsA2 and the loss of membrane PG is necessary and sufficient to produce high-level resistance to daptomycin in C. striatum. This elimination of PG and the absence of additional compensatory changes support the conclusion that PG is the target of daptomycin. This work highlights the importance of understanding how different bacterial species respond to lipopeptide antibiotics. Drugs that target membrane components may vary in efficacy by species due to differing abilities of species to alter or remove various membrane components. Strategies to prevent infection by multidrug-resistant organisms (MDROs) are scarce; however, autologous fecal microbiota transplantation (autoFMT) may limit gastrointestinal MDRO expansion. AutoFMT involves banking oneճ feces during a healthy state for later use in restoring gut microbiota following perturbation. In this pilot clinical trial involving 10 healthy participants, autoFMT was safe and well tolerated in the ten participants evaluated. The trial also evaluated the effects of amoxicillin-clavulanic acid (amox/clav) exposure and the ability of autoFMT to restore the microbiome. Both autoFMT and saline control restored metabolic capacity and resistance gene levels, but additional work is necessary to determine its ability to restore phylogeny. Importantly, metabolic capacity was perturbed following amox/clav even in cases where gross phylogeny remained unchanged

Impact of amoxicillin-clavulanate followed by autologous fecal microbiota transplantation on fecal microbiome structure and metabolic potential

The spread of multidrug resistance among pathogenic organisms threatens the efficacy of antimicrobial treatment options. The human gut serves as a reservoir for many drug-resistant organisms and their resistance genes, and perturbation of the gut microbiome by antimicrobial exposure can open metabolic niches to resistant pathogens. Once established in the gut, antimicrobial-resistant bacteria can persist even after antimicrobial exposure ceases. Strategies to prevent multidrug-resistant organism (MDRO) infections are scarce, but autologous fecal microbiota transplantation (autoFMT) may limit gastrointestinal MDRO expansion. AutoFMT involves banking one’s feces during a healthy state for later use in restoring gut microbiota following perturbation. This pilot study evaluated the effect of amoxicillin-clavulanic acid (Amox-Clav) exposure and autoFMT on gastrointestinal microbiome taxonomic composition, resistance gene content, and metabolic capacity. Importantly, we found that metabolic capacity was perturbed even in cases where gross phylogeny remained unchanged and that autoFMT was safe and well tolerated.Strategies to prevent multidrug-resistant organism (MDRO) infections are scarce, but autologous fecal microbiota transplantation (autoFMT) may limit gastrointestinal MDRO expansion. AutoFMT involves banking one’s feces during a healthy state for later use in restoring gut microbiota following perturbation. This pilot study evaluated the effect of autoFMT on gastrointestinal microbiome taxonomic composition, resistance gene content, and metabolic capacity after exposure to amoxicillin-clavulanic acid (Amox-Clav). Ten healthy participants were enrolled. All received 5 days of Amox-Clav. Half were randomized to autoFMT, derived from stool collected pre-antimicrobial exposure, by enema, and half to saline enema. Participants submitted stool samples pre- and post-Amox-Clav and enema and during a 90-day follow-up period. Shotgun metagenomic sequencing revealed taxonomic composition, resistance gene content, and metabolic capacity. Amox-Clav significantly altered gut taxonomic composition in all participants (n = 10, P  0.05, compared to enrollment). Alterations to microbial metabolic capacity occurred following antimicrobial exposure even in participants without substantial taxonomic disruption, potentially creating open niches for pathogen colonization. Our findings suggest that metabolic potential is an important consideration for complete assessment of antimicrobial impact on the microbiome. AutoFMT was well tolerated and may have contributed to phylogenetic recovery. (This study has been registered at ClinicalTrials.gov under identifier NCT02046525.

Use of in vitro human keratinocyte models to study the effect of cooling on chemotherapy drug-induced cytotoxicity

A highly distressing side-effect of cancer chemotherapy is chemotherapy-induced alopecia (CIA). Scalp cooling remains the only treatment for CIA, yet there is no experimental evidence to support the cytoprotective capacity of cooling. We have established a series of in vitro models for the culture of human keratinocytes under conditions where they adopt a basal, highly-proliferative phenotype thus resembling the rapidly-dividing sub-population of native hair-matrix keratinocytes. Using a panel of chemotherapy drugs routinely used clinically (docetaxel, doxorubicin and the active metabolite of cyclophosphamide 4-OH-CP), we demonstrate that although these drugs are highly-cytotoxic, cooling can markedly reduce or completely inhibit drug cytotoxicity, in agreement with clinical observations. By contrast, we show that cytotoxicity caused by specific combinatorial drug treatments cannot be adequately attenuated by cooling, supporting data showing that such treatments do not always respond well to cooling clinically. Importantly, we provide evidence that the choice of temperature may be critical in determining the efficacy of cooling in rescuing cells from drug-mediated toxicity. Therefore, despite their reductive nature, these in vitro models have provided experimental evidence for the clinically-reported cytoprotective role of cooling and represent useful tools for future studies on the molecular mechanisms of cooling-mediated cytoprotection

Microbiota restoration reduces antibiotic-resistant bacteria gut colonization in patients with recurrent Clostridioides difficile infection from the open-label PUNCH CD study

BACKGROUND: Once antibiotic-resistant bacteria become established within the gut microbiota, they can cause infections in the host and be transmitted to other people and the environment. Currently, there are no effective modalities for decreasing or preventing colonization by antibiotic-resistant bacteria. Intestinal microbiota restoration can prevent Clostridioides difficile infection (CDI) recurrences. Another potential application of microbiota restoration is suppression of non-C. difficile multidrug-resistant bacteria and overall decrease in the abundance of antibiotic resistance genes (the resistome) within the gut microbiota. This study characterizes the effects of RBX2660, a microbiota-based investigational therapeutic, on the composition and abundance of the gut microbiota and resistome, as well as multidrug-resistant organism carriage, after delivery to patients suffering from recurrent CDI. METHODS: An open-label, multi-center clinical trial in 11 centers in the USA for the safety and efficacy of RBX2660 on recurrent CDI was conducted. Fecal specimens from 29 of these subjects with recurrent CDI who received either one (N = 16) or two doses of RBX2660 (N = 13) were analyzed secondarily. Stool samples were collected prior to and at intervals up to 6 months post-therapy and analyzed in three ways: (1) 16S rRNA gene sequencing for microbiota taxonomic composition, (2) whole metagenome shotgun sequencing for functional pathways and antibiotic resistome content, and (3) selective and differential bacterial culturing followed by isolate genome sequencing to longitudinally track multidrug-resistant organisms. RESULTS: Successful prevention of CDI recurrence with RBX2660 correlated with taxonomic convergence of patient microbiota to the donor microbiota as measured by weighted UniFrac distance. RBX2660 dramatically reduced the abundance of antibiotic-resistant Enterobacteriaceae in the 2 months after administration. Fecal antibiotic resistance gene carriage decreased in direct relationship to the degree to which donor microbiota engrafted. CONCLUSIONS: Microbiota-based therapeutics reduce resistance gene abundance and resistant organisms in the recipient gut microbiome. This approach could potentially reduce the risk of infections caused by resistant organisms within the patient and the transfer of resistance genes or pathogens to others. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01925417 ; registered on August 19, 2013

The genetic architecture of the human cerebral cortex

The cerebral cortex underlies our complex cognitive capabilities, yet little is known about the specific genetic loci that influence human cortical structure. To identify genetic variants that affect cortical structure, we conducted a genome-wide association meta-analysis of brain magnetic resonance imaging data from 51,665 individuals. We analyzed the surface area and average thickness of the whole cortex and 34 regions with known functional specializations. We identified 199 significant loci and found significant enrichment for loci influencing total surface area within regulatory elements that are active during prenatal cortical development, supporting the radial unit hypothesis. Loci that affect regional surface area cluster near genes in Wnt signaling pathways, which influence progenitor expansion and areal identity. Variation in cortical structure is genetically correlated with cognitive function, Parkinson's disease, insomnia, depression, neuroticism, and attention deficit hyperactivity disorder

Blood flow and oxygenation in peritendinous tissue and calf muscle during dynamic exercise in humans

1. Circulation around tendons may act as a shunt for muscle during exercise. The perfusion and oxygenation of Achilles' peritendinous tissue was measured in parallel with that of calf muscle during exercise to determine (1) whether blood flow is restricted in peritendinous tissue during exercise, and (2) whether blood flow is coupled to oxidative metabolism. 2. Seven individuals performed dynamic plantar flexion from 1 to 9 W. Radial artery and popliteal venous blood were sampled for O(2), peritendinous blood flow was determined by (133)Xe-washout, calf blood flow by plethysmography, cardiac output by dye dilution, arterial pressure by an arterial catheter-transducer, and muscle and peritendinous O(2) saturation by spatially resolved spectroscopy (SRS). 3. Calf blood flow rose 20-fold with exercise, reaching 44 ± 7 ml (100 g)(−1) min(−1) (mean ± s.e.m.) at 9 W, while Achilles' peritendinous flow increased (7-fold) to 14 ± 4 ml (100 g)(−1) min(−1), which was 18 % of the maximal flow established during reactive hyperaemia. SRS-O(2) saturation fell both in muscle (from 66 ± 2 % at rest to 57 ± 3 %, P < 0.05) and in peritendinous regions (58 ± 4 to 52 ± 4 %, P < 0.05) during exercise along with a rise in leg vascular conductance and microvascular haemoglobin volume, despite elevated systemic vascular resistance. 4. The parallel rise in calf muscle and peritendinous blood flow and fall in O(2) saturation during exercise indicate that blood flow is coupled to oxidative metabolism in both tissue regions. Increased leg vascular conductance accompanied by elevated microvascular haemoglobin volume reflect vasodilatation in both muscle and peritendinous regions. However, peak exercise peritendinous blood flow reaches only ≈20 % of its maximal blood flow capacity