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W3109075414 abstract "Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract A key challenge in antibiotic stewardship is figuring out how to use antibiotics therapeutically without promoting the evolution of antibiotic resistance. Here, we demonstrate proof of concept for an adjunctive therapy that allows intravenous antibiotic treatment without driving the evolution and onward transmission of resistance. We repurposed the FDA-approved bile acid sequestrant cholestyramine, which we show binds the antibiotic daptomycin, as an ‘anti-antibiotic’ to disable systemically-administered daptomycin reaching the gut. We hypothesized that adjunctive cholestyramine could enable therapeutic daptomycin treatment in the bloodstream, while preventing transmissible resistance emergence in opportunistic pathogens colonizing the gastrointestinal tract. We tested this idea in a mouse model of Enterococcus faecium gastrointestinal tract colonization. In mice treated with daptomycin, adjunctive cholestyramine therapy reduced the fecal shedding of daptomycin-resistant E. faecium by up to 80-fold. These results provide proof of concept for an approach that could reduce the spread of antibiotic resistance for important hospital pathogens. eLife digest Antibiotics are essential for treating infections. But their use can inadvertently lead to the emergence of antibiotic-resistant bacteria that do not respond to antibiotic drugs, making infections with these bacteria difficult or impossible to treat. Finding ways to prevent antibiotic resistance is critical to preserving the effectiveness of antibiotics. Many bacteria that cause infections in hospitals live in the intestines, where they are harmless. But these bacteria can cause life-threatening infections when they get into the bloodstream. When patients with bloodstream infections receive antibiotics, the bacteria in their intestines are also exposed to the drugs. This can kill off all antibiotic-susceptible bacteria, leaving behind only bacteria that have mutations that allow them to survive the drugs. These drug-resistant bacteria can then spread to other patients causing hard-to-treat infections. To stop this cycle of antibiotic treatment and antibiotic resistance, Morley et al. tested whether giving a drug called cholestyramine with intravenous antibiotics could protect the gut bacteria. In the experiments, mice were treated systemically with an antibiotic called daptomycin, which caused the growth of daptomycin-resistant strains of bacteria in the mice’s intestines. In the laboratory, Morley et al. discovered that cholestyramine can inactivate daptomycin. Giving the mice cholestyramine and daptomycin together prevented the growth of antibiotic-resistant bacteria in the mice's intestines. Moreover, cholestyramine is taken orally and is not absorbed into the blood. It therefore only inactivates the antibiotic in the gut, but not in the blood. The experiments provide preliminary evidence that giving cholestyramine with antibiotics might help prevent the spread of drug resistance. Cholestyramine is already used to lower cholesterol levels in people. More studies are needed to determine if cholestyramine can protect gut bacteria and prevent antibiotic resistance in people. Introduction Vancomycin-resistant Enterococcus faecium (VR E. faecium) is an important cause of antibiotic-resistant infections in healthcare settings (Arias and Murray, 2012; García-Solache and Rice, 2019; O'Driscoll and Crank, 2015). The antibiotic daptomycin is one of the few remaining first-line therapies for VRE infection (O'Driscoll and Crank, 2015), but daptomycin-resistance is spreading in VRE populations (Judge et al., 2012; Kamboj et al., 2011; Kinnear et al., 2019; Woods et al., 2018). Therapeutic daptomycin use is thought to be a key driver of resistance (Kinnear et al., 2020; Woods et al., 2018). Managing the evolution of daptomycin-resistance in healthcare settings is crucial to future control of VRE infections. E. faecium is an opportunistic pathogen that colonizes the human GI tract asymptomatically, spreads via fecal-oral transmission, and causes symptomatic infections when introduced to sites like the bloodstream or the urinary tract (Arias and Murray, 2012). E. faecium colonizing the gut may be exposed to daptomycin during therapeutic use, potentially contributing to the transmission of daptomycin-resistant E. faecium. Daptomycin is administered intravenously to treat infections caused by pathogens including VRE and Staphylococcus aureus. Daptomycin is primarily eliminated by the kidneys, but 5–10% of the dose enters the intestines through biliary excretion (Woodworth et al., 1992). We hypothesize that this therapeutically unnecessary intestinal daptomycin exposure could drive resistance evolution in E. faecium colonizing the gut. Increased resistance in colonizing populations is important, because gut E. faecium populations are sources for nosocomial infections and transmission between patients (Alevizakos et al., 2017; Olivier et al., 2008). If unintended intestinal daptomycin exposure drives resistance evolution in E. faecium, this offers an opportunity to intervene. The opportunity emerges from a key feature of this system—the bacteria causing infection are physically separated from the population contributing to transmission. If daptomycin could be inactivated in the intestine without altering plasma concentrations, daptomycin could be used to kill bacteria at the target infection site without driving resistance in off-target populations. Preventing resistance evolution in these reservoir populations could protect patients from acquiring resistant infections, and it could limit the shedding of resistant strains and so onward transmission to other patients. We hypothesized that co-administering an oral adjuvant that reduces daptomycin activity would prevent selection for daptomycin-resistance in the gut during systemic daptomycin treatment. We tested this strategy using the adjuvant cholestyramine in a mouse VR E. faecium gut colonization model. Results Generation of daptomycin-resistant VR E. faecium in the mouse GI tract To directly test the proposition that systemic daptomycin treatment could select for resistance in the GI tract, and to generate daptomycin-resistant VR E. faecium mutants for subsequent experiments, we inoculated mice orally with daptomycin-susceptible VR E. faecium strains. Beginning one day after E. faecium inoculation, mice received daily doses of either subcutaneous daptomycin (50, 100, or 400 mg/kg), oral daptomycin (5, 50, 100, or 400 mg/kg), or a control mock injection for 5 days. We used a range of doses and routes of administration to maximize the likelihood of observing resistance emergence in at least one of the mice. The 50 and 100 mg/kg subcutaneous doses were selected to generate pharmacokinetics similar to clinical human doses (Mortin et al., 2007; Samonis et al., 2008), and the 5 mg/kg oral approximates the 5–10% of a daptomycin dose that is secreted into the intestines during standard intravenous treatment (Woodworth et al., 1992). We used two susceptible VR E. faecium strains, BL00239-1 (MICc = 2.0 (Minimum Inhibitory Concentration computed, see Methods)) and PR00708-14 (MICc = 2.7), which were originally isolated at the University of Michigan Hospital from a clinical bloodstream infection and a different patient’s clinical perirectal swab, respectively. Mouse fecal samples were collected to quantify VR E. faecium shedding and determine daptomycin susceptibility of isolated E. faecium clones. Only very high daptomycin doses (400 mg/kg subcutaneous, ≥50 mg/kg oral) consistently reduced fecal VR E. faecium below the level of detection during treatment; with lower doses, VR E. faecium shedding was often detectable throughout treatment (Figure 1A). For Strain BL00239-1, E. faecium clones with increased daptomycin-resistance were isolated from two of three mice following treatment with 100 mg/kg subcutaneous daptomycin (Figure 1B–C). We chose one of these daptomycin-resistant clones to use in subsequent experiments (strain BL00239-1-R, MICc = 8.6). Sequencing of the core genome showed that the resistant strain acquired a mutation in the major cardiolipin synthase clsA gene (R211L, CGA→CTA), which has been previously described in association with daptomycin-resistance (Adams et al., 2015), and a transposon insertion into the methionine sulfoxide reductase msrA gene (Zhao et al., 2010). For the second strain (PR00708-14), mice were treated with subcutaneous daptomycin (50, 100, or 200 mg/kg) or a mock injection. We screened for the emergence of increased resistance by plating mouse fecal suspensions on daptomycin-supplemented agar. Samples from two mice treated with 200 mg/kg daptomycin produced colonies on daptomycin-supplemented plates, and we isolated three clones from each of these samples. These isolated clones had increased daptomycin MICc relative to PR00708-14 by broth microdilution (MICc = 13.5, 11.5, and 8.0 from one mouse; MICc = 12.0, 29.8, and 12.0 from the second mouse).We chose one of these isolated clones for use in subsequent experiments (PR00708-14-R, MICc = 12.0). Genome sequencing revealed that PR00708-14 and PR00708-14-R differed by two mutations, which to our knowledge have not previously been associated with daptomycin-resistance. We identified mutations in a TerC family integral membrane protein (locus tag HMPREF0351_10759 in D0 E. faecium reference genome, I22T, CGA→CTA) and in a hypothetical protein (locus tag HMPREF0351_12146 in D0 E. faecium reference genome, frameshift, deleted G at 190nt). These experiments show that daptomycin-resistance can emerge de novo in E. faecium colonizing the GI tract following systemic daptomycin treatment. Figure 1 Download asset Open asset Emergence of daptomycin-resistant VR E. faecium in mouse GI tracts following subcutaneous daptomycin treatment. (A) VR E. faecium densities in fecal samples during and after daptomycin treatment (strain BL00239-1). Each line represents VR E. faecium densities from an individual mouse (N = 3 per treatment). The pink shaded region indicates days of daptomycin therapy. The dotted line marks the detection limit. Red dots indicate time points where clones were isolated for analysis shown in Panel B. The 400 mg/kg subcutaneous treatment was discontinued after 4 days due to apparent toxicity, and one mouse in this treatment was euthanized at Day 4. (B) Following daptomycin treatment, three VR E. faecium clones were isolated from the feces of each mouse. Filled points show the mean of triplicate daptomycin susceptibility (MICC) measurements for each clone, and open points show the individual measurements. Point shape indicates the mouse of origin. The dashed line marks the clinical breakpoint for daptomycin susceptibility. The ancestral clone (BL00239-1) used to inoculate mice is also shown. (C) For the 100 mg/kg subcutaneous treatment, VR E. faecium clones were isolated from each mouse at multiple time points. Filled points show the mean of triplicate daptomycin susceptibility (MICC) measurements for each clone, and open points show individual measurements. Color indicates mouse of origin. The dotted line marks the clinical breakpoint for daptomycin susceptibility. The resistant clone used in subsequent experiments (BL00239-1-R) is circled in red. Figure 1—source data 1 VR E. faecium fecal density data (Figure 1A). https://cdn.elifesciences.org/articles/58147/elife-58147-fig1-data1-v1.zip Download elife-58147-fig1-data1-v1.zip Figure 1—source data 2 MIC data (Figure 1B–C). https://cdn.elifesciences.org/articles/58147/elife-58147-fig1-data2-v1.zip Download elife-58147-fig1-data2-v1.zip Daptomycin treatment enriches for daptomycin-resistant VR E. faecium in the GI tract We used the de novo resistant mutants isolated above (Figure 1) to test whether daptomycin therapy selects for daptomycin-resistance in intestinal VR E. faecium populations when a resistant mutant is already present. We orally inoculated mice with a 1:20 mixture of the experimentally generated daptomycin-resistant and susceptible VR E. faecium strains (BL00239-1-R and BL00239-1). Note that this approach – seeding inocula with known numbers of resistant bacteria – allows the response to selection to be measured while avoiding the experimental noise introduced by mutation waiting times. Mice were treated with subcutaneous daptomycin (50, 75, 100, or 200 mg/kg) for five or ten days after VRE inoculation. Control mice received either a mock saline injection or no injection. Fecal samples from Days 8 and 14 post-inoculation were plated in triplicate to quantify total VR E. faecium density, and samples were also plated on daptomycin-supplemented agar to estimate the proportion of VR E. faecium that were daptomycin-resistant. Control populations remained susceptible to daptomycin, but all doses and durations of daptomycin dramatically enriched for resistance in the GI tract (Figure 2A–B). At both time points, controls had significantly lower proportions of resistant bacteria than daptomycin-treated mice (Figure 2A–B; mixed effects negative binomial regression, p<0.01, see Model one in Supplementary file 1). The effect sizes (Cohen’s d) at Days 8 and 14 were 5.90 (95% CI 4.94, 6.86) and 2.34 (95% CI 1.82, 2.87), respectively. The absolute numbers of VRE enumerated in fecal samples did not vary significantly between treatments (mixed effects negative binomial regression, Model two in Supplementary file 1; Figure 2—figure supplement 1). Figure 2 with 1 supplement see all Download asset Open asset Subcutaneous daptomycin treatment selects for resistance in the GI tract. (A-B) Mouse fecal suspensions were plated on Enterococcus-selective plates with daptomycin (+ DAP) and without daptomycin (- DAP) at Day 8 (A) and Day 14 (B). Each filled point represents the mean of triplicate measures from a single mouse sample, and open points show individual measurements. Mice were treated with daptomycin for 5 days (triangles) or 10 days (squares) at the doses listed at times denoted (gray crosses in C; N = 5 mice per treatment). Samples with VR E. faecium density <3×103 CFU/10 mg feces were not plated for this assay due to insufficient bacterial density. (C) Recovered fecal daptomycin measured by LC-MS for a subset of mice. Each line tracks daptomycin measurements from a single mouse sampled at Days 2, 6, and 8. (D) For the subset of fecal samples analyzed by LC-MS, fecal daptomycin plotted against fecal VR E. faecium densities (samples from all available treatments and time points plotted together). Dotted line indicates the MIC of the susceptible strain BL00239-1 (MIC = 2 µg/mL or 2 µg/g). Figure 2—source data 1 VR E. faecium daptomycin susceptibility data (Figure 2A–B). https://cdn.elifesciences.org/articles/58147/elife-58147-fig2-data1-v1.zip Download elife-58147-fig2-data1-v1.zip Figure 2—source data 2 Fecal daptomycin concentration data (Figure 2C–D). https://cdn.elifesciences.org/articles/58147/elife-58147-fig2-data2-v1.zip Download elife-58147-fig2-data2-v1.zip The dramatic enrichment for daptomycin-resistant VR E. faecium in treated mice showed that subcutaneously-administered daptomycin produced GI tract concentrations high enough to select for resistance. To quantify fecal daptomycin concentrations, we analyzed fecal samples from a subset of daptomycin-treated mice by liquid chromatography-mass spectrometry (LC-MS) (Figure 2C). Samples from all time points tested (Days 2, 6, and 8) contained detectable daptomycin, and concentrations generally peaked at the end of treatment (Day 6). Higher daptomycin doses generally corresponded to higher fecal concentrations, but concentrations were highly variable and overlapped between treatments. While fecal VR E. faecium densities correlated poorly with the daptomycin dose administered (Figure 2—figure supplement 1), fecal VR E. faecium densities correlated with the amount of daptomycin recovered in feces (Figure 2D). These data confirmed that subcutaneously-administered daptomycin at our experimental doses generated a range of daptomycin concentrations in the GI tract that included inhibitory concentrations for the susceptible VR E. faecium strain. We ran two additional experiments to further investigate the competitive dynamics between this susceptible and resistant pair (BL00239-1 and BL00239-1-R) in the presence and absence of daptomycin treatment. First, we tested whether susceptible bacteria competitively suppressed resistant bacteria in the GI tract. We inoculated mice with either a mixture of 108 CFU susceptible + 103 CFU-resistant VR E. faecium, or with a resistant-only inoculum at one of two inoculum sizes (108 CFU or 103 CFU). Mice received 5 days of subcutaneous daptomycin injections at 100 mg/kg or control saline injections. Shedding of resistant and susceptible bacteria were quantified at time points throughout the experiment by plating (Figure 3A). In the absence of daptomycin treatment, the daptomycin-susceptible strain remained the most prevalent in mixed populations. When mixed populations were exposed to daptomycin, resistance increased to high frequency in three populations, and the population size fell dramatically in the remaining two populations. In mice inoculated with only 103 CFU-resistant bacteria, the resistant clone was able to grow to high numbers with or without daptomycin. These data were consistent with the competitive suppression of the resistant strain by the susceptible strain in the absence of daptomycin treatment, and competitive release of the resistant strain during daptomycin treatment (Day et al., 2015; Wargo et al., 2007). Figure 3 Download asset Open asset Competitive dynamics between daptomycin-resistant and susceptible VRE faecium. (A) Each panel shows VR E. faecium counts on plates with daptomycin (+DAP, red line) and without daptomycin (-DAP, blue line) for a single mouse over time. Labels show initial inocula. Red shading indicates days of daptomycin treatment (100 mg/kg daily subcutaneous injections). (B) In a second experiment, mice were inoculated with a mix of susceptible and resistant bacteria (S+R, 20% R), resistant bacteria only (R), or susceptible bacteria only (S). Mice received no drug treatment. At Day 14, fecal suspensions were plated on plates with daptomycin (+DAP) and without daptomycin (-DAP) to determine whether the resistant strain had decreased in frequency. The starting inoculum dose is shown in red, and Day 14 samples from each mouse are shown in gray. Filled points show means of replicate measurements for a sample, and open circles show individual measurements. Note that resistant bacteria do not form colonies at 100% efficiency on +DAP plates. Figure 3—source data 1 VR E. faecium density data (Figure 3A). https://cdn.elifesciences.org/articles/58147/elife-58147-fig3-data1-v1.zip Download elife-58147-fig3-data1-v1.zip Figure 3—source data 2 VR E. faecium daptomycin susceptibility data (Figure 3B). https://cdn.elifesciences.org/articles/58147/elife-58147-fig3-data2-v1.zip Download elife-58147-fig3-data2-v1.zip Next, we tested whether the frequency of daptomycin-resistant VR E. faecium would decrease over time in the absence of daptomycin treatment, potentially indicating that daptomycin-resistance comes at a fitness cost. We inoculated mice with a 1:5 mixture of daptomycin-resistant and susceptible VR E. faecium. Mice received no daptomycin treatment. After 14 days, the proportion of resistant bacteria had declined (one sample t-test, t = −22.42, df = 9, p<0.01, Cohen’s d = 7.09), consistent with a competitive disadvantage (fitness cost) to the daptomycin-resistance mutation (Figure 3B). Control mice inoculated with only resistant or only susceptible bacteria did not have significantly different proportions of resistance between days 0 and 14 (resistant: t = −3.73, df = 2, p=0.06; susceptible: t = 1, df = 2, p=0.42). In vitro characterization of cholestyramine as a potential adjuvant If an orally-administered adjuvant could reduce daptomycin activity in the GI tract, this could prevent the emergence of daptomycin-resistant E. faecium in the gut, potentially reducing transmission of resistant bacteria without impacting the effectiveness of intravenous daptomycin therapy. We identified cholestyramine, an FDA-approved bile-acid sequestrant, as a potential adjuvant for daptomycin therapy. Cholestyramine is a high-molecular weight anion exchange resin that binds with bile acids, forming an insoluble complex that is excreted in the feces (Jacobson et al., 2007). Cholestyramine is known to interact with a number of co-administered drugs through the same mechanism, reducing their bioactivity (Jacobson et al., 2007). We hypothesized that cholestyramine would bind daptomycin based on their chemical structures. In vitro tests were consistent with cholestyramine binding daptomycin. Daptomycin solutions were incubated with cholestyramine, and then the cholestyramine was removed by centrifugation. The resulting supernatants were analyzed for changes in daptomycin concentration and activity. Daptomycin concentrations can be measured directly by ultraviolet (UV) absorbance at 364 nm (Figure 4A). Daptomycin concentrations were reduced in supernatants after incubation with cholestyramine in a dose-dependent manner (Figure 4B). Additionally, daptomycin solutions incubated with cholestyramine had reduced antibiotic activity against E. faecium in broth microdilution (Figure 4C). Together, these results were consistent with cholestyramine removing daptomycin from solution, supporting cholestyramine as a candidate adjuvant for daptomycin therapy. Figure 4 Download asset Open asset Cholestyramine captures daptomycin in vitro. (A) Calibration curve (best fit linear regression) showing that daptomycin concentration can be measured by UV absorption at 364 nm (N = 3 per concentration). (B) Daptomycin concentration was reduced in solutions treated with cholestyramine (N = 3 per concentration). (C) Daptomycin solutions treated with cholestyramine had reduced biological activity against VRE in broth microdilutions (N = 3 per antibiotic treatment). Bacterial densities (OD600) following growth in the presence of daptomycin (DAP), daptomycin solution treated with cholestyramine (DAP + CHOL), or saline solution treated with cholestyramine (saline + CHOL) are shown. Concentrations are shown as the initial concentration of daptomycin in solution prior to cholestyramine treatment. Saline controls were constant across all listed concentrations. Horizontal line shows detection threshold. Figure 4—source data 1 Calibration curve data (Figure 4A). https://cdn.elifesciences.org/articles/58147/elife-58147-fig4-data1-v1.zip Download elife-58147-fig4-data1-v1.zip Figure 4—source data 2 Daptomycin concentration data (Figure 4B). https://cdn.elifesciences.org/articles/58147/elife-58147-fig4-data2-v1.zip Download elife-58147-fig4-data2-v1.zip Figure 4—source data 3 Broth microdilution data, OD600 readings from 96-well plate (Figure 4C). https://cdn.elifesciences.org/articles/58147/elife-58147-fig4-data3-v1.zip Download elife-58147-fig4-data3-v1.zip Adjunctive cholestyramine therapy prevents emergence of daptomycin-resistance We conducted four experiments to test whether adjunctive therapy with cholestyramine could prevent the emergence of daptomycin-resistant VR E. faecium in the mouse GI tract. In each experiment, mice were orally inoculated with a 1:20 mixture of daptomycin-resistant and susceptible VR E. faecium and then treated with subcutaneous daptomycin injections for 5 days. Densities of total VR E. faecium and daptomycin-resistant VR E. faecium were determined by plating (Figure 5, Figure 5—figure supplements 1–5). The experiments tested the evolutionary impact of oral cholestyramine in different mouse strains, with different VR E. faecium strains, and with different timing of cholestyramine administration. The design of the four experiments was as follows: (A) Swiss Webster mice with E. faecium strains BL00239-1 and BL00239-1-R, with cholestyramine started one day before daptomycin (Figure 5—figure supplement 1), (B) C57BL/6 mice with E. faecium strains BL00239-1 and BL00239-1-R, with cholestyramine started one day before daptomycin (Figure 5—figure supplement 2), (C) Swiss Webster mice with E. faecium strains PR00708-14 and PR00708-14-R, with cholestyramine started one day before daptomycin (Figure 5—figure supplement 3), and (D) Swiss Webster mice with E. faecium strains BL00239-1 and BL00239-1-R, with cholestyramine started the same day as daptomycin (Figure 5—figure supplement 4). Data from these experiments were analyzed together, with a block effect included in the models. Because bacterial densities were found not to correlate to daptomycin dose (Figure 2—figure supplement 1), all daptomycin doses were combined into a single group for analysis. Figure 5—figure supplements 1–4 show these data broken down by experiment and by daptomycin dose. For daptomycin-treated mice shedding detectable levels of VR E. faecium by our plating assay (at least 20 CFU/10 mg feces at a given time point), the cholestyramine-supplemented diet reduced the proportion of daptomycin-resistant VR E. faecium (mixed effects binomial regression, p<0.01, Model three in Supplementary file 1). The effect size (Cohen’s d) of cholestyramine diet on the proportion of resistant bacteria in daptomycin-treated mice was 0.41 (95% CI 0.01, 0.82) at Day 2, 0.56 (95% CI 0.13, 1.00) at Day 4, 0.90 (95% CI 0.45, 1.35) at Day 6, 1.08 (95% CI 0.63, 1.53) at Day 8, and 0.63 (95% CI 0.21, 1.05) at Day 14. In addition, to more accurately determine resistance proportions for Days 8 and 14, we plated an estimated 200 CFU from each sample in triplicate on plates with and without daptomycin (Figure 5—figure supplement 5). This second assay confirmed that cholestyramine reduced the proportion of resistant VR E. faecium at these time points (mixed effects binomial regression, p<0.01, Model four in Supplementary file 1; Cohen’s d 1.25 (95% CI 0.97, 1.53) at Day 8, 0.73 (95% CI 0.48, 0.98) at Day 14). Figure 5 with 5 supplements see all Download asset Open asset Adjunctive cholestyramine reduces enrichment of daptomycin-resistance in the GI tract. (A) The proportion of fecal VR E. faecium that were daptomycin-resistant over time in mice. Proportions were determined by plating on agar with daptomycin (+DAP) and without daptomycin (-DAP). Data shown were combined from four experiments (for each diet N = 20 controls and N = 50 daptomycin-treated, mean + SEM shown). Proportions were not determined for samples with <20 CFU VR E. faecium per 10 mg feces, as these densities were below the limit of detection for this plating assay, and these samples were not included in Panel A. The pink shaded region indicates days of daptomycin therapy. (B) Total VR E. faecium densities corresponding to data shown in Panel A (for each diet N = 20 controls and N = 50 daptomycin-treated, mean + SEM shown). Dotted line shows total density (-DAP) and solid line shows the density of daptomycin-resistant VR E. faecium (+DAP). All samples, including those with low densities, were included in Panel B. Figure 5—source data 1 VR E. faecium fecal density data. https://cdn.elifesciences.org/articles/58147/elife-58147-fig5-data1-v1.zip Download elife-58147-fig5-data1-v1.zip We also quantified absolute densities of daptomycin-resistant and susceptible VR E. faecium over time by plating samples from Days 0, 1, 2, 4, 6, 8, and 14 (Figure 5). These data showed that the cholestyramine-supplemented diet reduced fecal shedding of daptomycin-resistant VR E. faecium in daptomycin-treated mice (Antibiotic*Diet*Day interaction p<0.01, Model five in Supplementary file 1). The effect size was greatest in the days after daptomycin treatment. The effect size (Cohen’s d) of cholestyramine diet on shedding of resistant bacteria in daptomycin-treated mice was 0.43 (95% CI 0.02, 0.83) at Day 2, 0.08 (95% CI −0.32, 0.48) at Day 4, 0.12 (95% CI −0.28, 0.51) at Day 6, 0.36 (95% CI −0.04, 0.76) at Day 8, and 0.57 (95% CI 0.16, 0.98) at Day 14. Total VR E. faecium shedding was also influenced by the addition of cholestyramine (Antibiotic*Diet*Day interaction p<0.01, Model six in Supplementary file 1). Here the effect size is greatest during daptomycin treatment. The effect size (Cohen’s d) of cholestyramine diet on total shedding in daptomycin-treated mice was 0.84 (95% CI 0.43, 1.23) at Day 2, 0.36 (95% CI −0.04, 0.76) at Day 4, 0.31 (95% CI −0.09, 0.71) at Day 6, 0.04 (95% CI −0.36, 0.43) at Day 8, and 0.40 (95% CI 0.00, 0.80) at Day 14. If we consider only the control treatments, where there is no possibility of cholestyramine protecting against daptomycin killing, the addition of cholestyramine to the diet does not significantly influence total VR E. faecium counts alone (p=0.63, Model seven in Supplementary file 1) or in combination with day (p=0.18). Discussion Here we have shown proof of concept for an adjunctive therapy approach to prevent the emergence of daptomycin-resistant E. faecium in the GI tract during daptomycin therapy. Ideally, this approach would allow clinicians to treat bloodstream infections with intravenous daptomycin without fueling the hospital transmission of multidrug-resistant bacteria. This would be a novel approach because the desired outcome is reduced resistance evolution and reduced transmission of resistant pathogens. Colonization with VR E. faecium" @default.
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W3109075414 title "Author response: An adjunctive therapy administered with an antibiotic prevents enrichment of antibiotic-resistant clones of a colonizing opportunistic pathogen" @default.
W3109075414 doi "https://doi.org/10.7554/elife.58147.sa2" @default.
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