Process for reduction of drug-resistant bacteria

Abstract

A process and system for removal of antibacterial compounds from a patient's body can include having a patient digest a polymeric material. The consumed material can pass through the patient's digestive system to remove antibacterial material that may reside therein as a consequence of an antibacterial treatment provided to the patient. Removal of the antibacterial material from the patient's digestive system can avoid evolution of antimicrobial resistance. In some embodiments, the polymeric material can include sevelamer (SEV) or other suitable polymeric material.

Description

PROCESS FOR REDUCTION OF DRUG-RESISTANT BACTERIACROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Patent Application No.63 / 627,259, filed on January 31, 2024, the entire contents of which is incorporated by reference, and to U.S. Provisional Patent Application No. 63 / 647,745, filed on May 15, 2024, the entire contents of which is incorporated by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH DEVELOPMENT

[0002] This invention was made with government support under Grant No.NI23HFPXXXXXG054 awarded by the United States Department of Agriculture. The Government has certain rights in the invention.FIELD

[0003] Embodiments relate to processes and apparatuses configured to reduce, hinder, or avoid the growth or development of drug resistant bacteria. Embodiments can be adapted to help block or avoid bacterial growth within the body of an animal or a human, for example. Embodiments can be adapted to help protect microbiome within the body of an animal or a human.BACKGROUND

[0004] Vancomycin (VAN) and daptomycin (DAP) are often utilized as last-line antibiotics to treat multi drug-resistant bacterial infections (e.g. Gram-positive Enterococcus infections).VAN is a glycopeptide antibiotic, which is synthesized by soil-dwelling Amycolatopsis orientalis through fermentation. VAN blocks bacterial cell wall biosynthesis via binding the aglycon moiety to the D-alanyl-D-alanine (DAD A) dipeptide, located in the peptidoglycan layerof cell wall. This binding occurs via forming five hydrogen bonds between VAN heptapeptide backbone and the DADA.

[0006] DAP is a lipopeptide antibiotic, which is synthesized by Streptomyces roseosporus in decanoic acid-enriched media via fermentation. It is an amphiphilic molecule with a hydrophilic head decorated with 13 amino acids linked to a lipophilic tail consisting of a decanoyl fatty acid. DAP antibacterial activity is attributed to its binding to the bacteria cell membrane, resulting in cell death. Hydrophobic interactions between DAP lipid tail and cell membrane phospholipid bilayers allow DAP to penetrate and depolarize the cell, damaging the intracellular components.

[0007] Patients with are frequently treated with I.V. V AN for prophylaxis or treatment of gram positive bacteria. If they have VAN-resistant Enterococcus faecium (VREfrri) or other infections, they may be treated with DAP. In the event the bacterial infection is resistant to VAN or DAP, it may be difficult to treat the infection (c.g. there may not be a defined standard of care for treating such a condition).SUMMARY

[0008] In clinical settings, VAN and DAP are typically administered intravenously (IV), and their excess amount in the bloodstream is mainly eliminated via urine. However, 5 - 10 % of the IV dose can enter the gastrointestinal (GI) tract via biliary excretion wherein it not only has no therapeutic value but also drives the resistance evolution in natural harmless bacteria within the gut of a patient (e.g. E. faecium colonizers). We believe that this type of drug resistance evolution in natural, harmless bacteria can grow the threat of antimicrobial resistance (AMR).

[0009] We have determined that removing VAN and DAP from the intestines of a patient can avoid hindering their IV effectiveness as a treatment for a bacterial infection and can also enableantibiotic use without driving resistance in the GI tract of the patient. To date, a few adjuvants have been used to remove antibiotics from the GI tract, including activated charcoal, 0-lactamase inhibitors, DADA-mimicking peptides, and cholestyramine.

[0010] Activated charcoal removes a wide spectrum of antibiotics from the gut, but does not interact with VAN. P-lactamases enzymatically deactivate the [J-lactam antibiotic classes in the GI tract; however, they are not effective against non-fl-lactam VAN and DAP antibiotics. Peptide analogs are theoretically able to adsorb VAN, but the peptide analogs rapidly degrade upon contact with digestive enzymes that can significantly impede their in vivo use and are therefore not expected to be effective in the GI tract of an animal (e.g. a human).

[0011] In Morley, V.J., Kinnear, C.L., Sim, D.G., Olson, S.N., Jackson, L.M., Hansen, E., Usher, G.A., Showalter, S.A., Pai, M.P., Woods, R.J., & Read, A.F. (2020). Az? adjunctive therapy approach prevents antibiotic resistance emergence in opportunistic pathogens colonizing the gut. eLife 9:e58147 and Morley, V.J., Sim, D.G., Penkevitch, A., Woods, R.J., & Read, A.F. (2022). An orally administered drug prevents selection for antibiotic-resistant bacteria in the gut during daptomycin therapy. Evolution, Public Health and Medicine 10: 439- 446, it was demonstrated that IV DAP injected mice that were orally administered with cholestyramine, a bile acid sequestrant, can undergo a significant decrease in the enrichment and shedding of DAP -resistant bacteria (e.g. VRE / m). In some embodiments, an 80-fold reduction can be provided without undergoing any de novo resistance mutations. We also investigated the mechanism via which the oral adjuvant prevented GI DAP activity, showing that the electrostatic binding of self-assembled DAP to cholestyramine can facilitate DAP removal from a patient’s GI. See e.g. Yeh, S. L.; et aL, A. Ion Exchange Biomaterials to Capture Daptomycin and PreventResistance Evolution in Off-Target Bacterial Populations. ACS Appl Mater Interfaces 2022, 14(38), 42864-42875 (https: / / doi.org / 10.1021 / acsami.2cl4894). Despite its initial success in removing DAP, cholestyramine was unsuccessful in VAN removal.

[0012] We have found, however, that the anti-antibiotic capability of sevelamer (SEV), which is a Federal Drug Administration (FDA) approved weakly basic anion-exchange polymer, can provide antibiotic drug removal from the GI of a patient for DAP as well as VAN. SEV can include polyallylamine that is crosslinked with epichlorohydrin. SEV can be a partial hydrochloride salt that has approximately 40% amine hydrochloride and 60% sevelamer base in some configurations. The amine groups of sevelamer can become partially protonated in the intestine and can interact with phosphate ions through ionic bonding and / or hydrogen bonding.

[0013] SEV is conventionally used to treat hyperphosphatemia via binding phosphate in the GI tract, lowering the phosphate levels that otherwise causes serious complications in kidney patients. However, we have surprisingly found that SEV may be able to be provided to patients to provide an SEV -mediated VAN removal to help avoid or reduce the growth of drug-resistant bacteria within the GI of a patient and / or protect microbiome. We also believe such treatments can remove DAP as well as VAN. We also confirmed that such an SEV treatment can provide this type of effect for at least VAN removal by conducting in vitro experiments at controlled initial antibiotic concentrations, pH, and ionic strengths. Such confirmation work includes the discussion of experimentation discussed herein. That experimentation work includes the examination of the removal of antibiotics via SEV that we examined under the influence of individual intestinal fluid components, e.g., bile acid (BA), maleic acid (MA), phosphatidylcholine (PC), and the whole simulated intestinal fluid (SIF). The SEV-mediatedantibiotic removal was also assessed via a standard antibacterial microdilution assay againstVSE / / 77 and VRE / zu, and its efficacy in some embodiments were also assessed in mouse models of Enterococcus faecium intestinal colonization.

[0014] We have surprisingly found that embodiments of our process for use of SEV can be able to significantly reduce AMR that can be induced by VAN and / or DAP treatments in patients. Embodiments of our process and systems configured to utilize an embodiment of our process can permit the implementation of strategies to use antibiotics to treat an infection affecting a patient without contributing to the promotion of the growth of drug resistant bacteria. We believe that embodiments of our process for treating a patient with SEV to reduce or remove the VAN and / or DAP in their GI can be utilized to implement an embodiment of our process can help improve the control of hard-to-treat infections in healthcare settings by reducing the rate of growth and the development rate of drug resistant bacteria and the evolution of such bacteria.

[0015] SEV is just one example of other polymeric materials that can be consumed by a patient to remove VAN from the patient’s intestines. Embodiments can be adapted so that both VAN and DAP can be effectively removed from the patient’s GI tract (e.g. by eliminating the bioreactivity of VAN and / or DAP material within the patient’s intestinal tract, or GI tract, by substantially reducing the bioreactivity of VAN and / or DAP material within the patient’s intestine, etc.). Removal of VAN (or a combination of VAN and DAP) or removal or substantial reduction of at least the bioreactivity of VAN or a combination of VAN and DAN in a patient’s GI tract can have various different benefits in addition to helping to prevent AMR evolution in bacteria. For example, the removal of the VAN and / or DAP (or at least the bioreactivity of VAN and / or DAP) can help avoid the antibacterial agents negatively affecting a patient’s gut biome(e.g. by destroying or killing some of the patient’s beneficial gut bacteria, etc.). For example, embodiments can permit a patient who is receiving VAN, DAP, and / or other antibiotic (e.g. linezolid) to avoid microbiome dysbiosis, which can be associated with many metabolic, neurological and immune -mediated diseases, as well as recurrent C. difficile infections.

[0016] The antibiotic removal agent or antibiotic deactivation agent that may be used in embodiments of our process to remove the bioreactivity of VAN from a patient’s GI tract and / or remove the bioreactivity of VAN as well as DAP and / or other antibacterial agents (e.g. linezolid), can utilize other compounds in addition to or as an alternative to SEV. For example, the antibacterial agent removal compound or the antibiotic deactivation agent to be ingested by a patient undergoing a VAN treatment (e.g. IV VAN treatment, treatment of VAN in combination with DAP, etc.) can utilize one or more ion exchange resins (e.g. at least one anion exchange polymeric material, at least one cation exchange polymeric material, at least one polymeric material, etc.). Examples of some cation exchanger polymeric material options can include Sulfonic acid functional groups such as, for example, Dowex 50 and Amberlite IR-120, and weak Acidic cation exchange resins having carboxylic acid functional groups such as e.g., Amberlite IRC-50, and Dowex MAC. Examples of anion exchanger polymeric materials can include anion exchange resins such as, for example, Amberlite IRA-400 and Dowex 1X8 as well as Amberlite IRA-67 and Dowex MWA-1. Mixed bed resins can also be utilized (e.g. Chelex 100) and / or selective ion exchange resins can be used, such as, e.g., Permutit and Amberlite CG- 50 for nitrate removal. Specialty resins or mixed material resins can also be other examples of suitable polymeric material options. As yet other example, gel-type resins or porous resins may be utilized.

[0017] For example, one or more of the following can be ingested by a patient as an antibacterial agent removal compound or as an antibiotic deactivation agent: Veltassa (generic name: patiromer), Kionex (generic name: sodium polystyrene sulfonate), Lokelma (generic name: sodium zirconium cyclosilicate), Kayexalate (generic name: sodium polystyrene sulfonate), Kalexate (generic name: sodium polystyrene sulfonate), Colestipol (sold under the trade names Colestid and Cholestabyl), Tolevamer (sold under trade names: Kayexalate, Kionex, Resonium Calcium, and Solystat), Polacrilex resin (Amberlite IRP64), Polacrilin potassium (Amberlite IRP88), WelChol (colesevelam), SEV, magnesium-containing compounds, calcium-containing compounds, iron-containing compounds, zinc-containing compounds, antacids containing aluminum or magnesium, proton pump inhibitors, or combinations of these materials. We believe these types of polymers can adsorb VAN, DAP, and / or other antibiotics in the GI tract of a patient to protect antibiotic sensitive bacteria and prevent the upselection for antibacterial agent resistance in the GI microbiome of the patient.

[0018] VAN is frequently the drug of choice for treating infections caused by Methicillin Resistant Staphylococcus aureus. Vancomycin-resistant MRS A (VR-MRSA or VRSA) has been sporadically reported but while it is still rare, it is increasing globally. A vancomycin-inhibitor can substantially reduce the probability that VRSA could arise, first by reducing the number of VRE populations which could transfer resistance via horizontal transfer to MRSA, and second by reducing the selection for VRSA that has arisen. Embodiments of our process can help provide VAN inhibition in a patient’s GI tract to help avoid formation of VRSA while also providing other benefits as discussed above, for example.

[0019] In an exemplary embodiment, a process for removing one or more antibacterial compounds from a gastrointestinal (GI) tract of a patient comprises administering at least one antibiotic removal agent or at least one antibiotic deactivation agent to the patient for digestion so that the at least one antibiotic removal agent or the at least one antibiotic deactivation agent is present in the GI tract of the patient; the at least one antibiotic removal agent or the or at least one antibiotic deactivation agent effectively removing the one or more antibacterial compounds present in the GI tract.

[0020] In some embodiments, the one or more antibacterial compounds include Vancomycin (VAN).

[0021] In some embodiments, the one or more antibacterial compound also includes daptomycin (DAP) and / or linezolid.

[0022] In some embodiments, the at least one antibiotic removal agent or the or at least one antibiotic deactivation agent effectively removing the one or more antibacterial compounds present in the GI tract includes the at least one antibiotic removal agent interacting with the one or more antibacterial compounds present in the GI tract such that one or more antibacterial compounds present in the GI tract are non-bioreactive with bacteria within the GI tract.

[0023] In some embodiments, the at least one antibiotic removal agent or the at least one antibiotic deactivation agent is comprised of sevelamer (SEV).

[0024] In some embodiments, the at least one antibiotic removal agent or the at least one antibiotic deactivation agent is comprised of patiromer, sodium polystyrene sulfonate, sodium zirconium cyclosilicate, sodium polystyrene sulfonate, sodium polystyrene sulfonate, colestipol, tolevamer, polacrilex resin (Amberlite IRP64), polacrilin potassium (Amberlite IRP88),colesevelam, sevelamer (SEV), magnesium-containing compounds, calcium-containing compounds, iron-containing compounds, zinc-containing compounds, antacids containing aluminum or magnesium, proton pump inhibitors, or combinations thereof.

[0025] In some embodiments, the process comprises treating the patient with the one or more antibacterial compounds to treat an infectious disease in the patient.

[0026] In some embodiments, the infectious disease is a bacterial infection that is not located in the GI tract.

[0027] In some embodiments, the administering of the at least one antibiotic removal agent or the at least one antibiotic deactivation agent to the patient for the patient to digest so that the at least one antibiotic removal agent or the at least one antibiotic deactivation agent is present in the GI tract of the patient comprises feeding the patient the at least one antibiotic removal agent or the at least one antibiotic deactivation agent such that the at least one antibiotic removal agent or the at least one antibiotic deactivation agent is orally consumed by the patient.

[0028] In some embodiments, the administering of the at least one antibiotic removal agent or the at least one antibiotic deactivation agent to the patient for the patient to digest so that the at least one antibiotic removal agent or the at least one antibiotic deactivation agent is present in the GI tract of the patient comprises feeding the patient the at least one antibiotic removal agent or the at least one antibiotic deactivation agent periodically while the patient undergoes a treatment for an infection that includes multiple injections of the one or more antibacterial compounds into the patient at different spaced apart intervals.

[0029] In some embodiments, the administering is performed periodically different times a day for a number of days.

[0030] In some embodiments, the at least one antibiotic removal agent or the at least one antibiotic deactivation agent effectively removes the one or more antibacterial compounds present in the GI tract such that bacteria within the GI tract is unaffected by the antibacterial compounds.

[0031] In some embodiments, the process is performed in a hospital, a care facility, or a farm.

[0032] In some embodiments, the process comprises injecting the patient with and / or orally administering to the patient the one or more antibacterial compounds to treat an infectious disease in the patient.

[0033] In some embodiments, the process comprises injecting the patient with the one or more antibacterial compounds to treat an infectious disease in the patient; wherein the infectious disease is a bacterial infection that is not located in the GI tract; and wherein the administering of the at least one antibiotic removal agent or the at least one antibiotic deactivation agent to the patient for the patient to digest so that the at least one antibiotic removal agent or the at least one antibiotic deactivation agent is present in the GI tract of the patient comprises feeding the patient the at least one antibiotic removal agent or the at least one antibiotic deactivation agent such that the at least one antibiotic removal agent is orally consumed by the patient.

[0034] In some embodiments, the at least one antibiotic removal agent or the at least one antibiotic deactivation agent is comprised of a polymeric material.

[0035] In some embodiments, the at least one antibiotic removal agent or the at least one antibiotic deactivation agent is comprised of at least one anion exchange polymeric material.

[0036] In some embodiments, the at least one antibiotic removal agent or the at least one antibiotic deactivation agent is comprised of at least one cation exchange polymeric material.

[0037] In some embodiments, the at least one antibiotic removal agent or the at least one antibiotic deactivation agent is comprised of a nanoparticle and / or microparticle.

[0038] In some embodiments, the at least one antibiotic removal agent or the at least one antibiotic deactivation agent is comprised of a small molecule.

[0039] In some embodiments, the at least one antibiotic removal agent or the at least one antibiotic deactivation agent is comprised of an organic and / or inorganic material.

[0040] In some embodiments, the at least one antibiotic removal agent or the or at least one antibiotic deactivation agent is configured protect microbiome present in the G1 tract of the patient.

[0041] Further features, aspects, objects, advantages, and possible applications of the present invention will become apparent from a study of the exemplary embodiments and examples described below, in combination with the Figures, and the appended claims.BRIEF DESCRIPTION OF THE FIGURES

[0042] The above and other objects, aspects, features, advantages, and possible applications of the present invention will be more apparent from the following more particular description thereof, presented in conjunction with the following drawings. It should be understood that like reference numbers used in the drawings may identify like components.

[0043] FIG. 1 is a schematic illustration of an exemplary embodiment of our process for removal of antibiotic compounds from the body of a patient, which can help prevent AMR. In the exemplary embodiment shown in FIG. 1, SEV can be orally consumed by a patient for being digested by the patient so that the SEV captures Gl-excreted VAN or DAP, disabling the off-target antibiotics from reaching the colonizing opportunistic bacteria in the intestines for preventing antibiotic resistance evolution in susceptible bacteria.

[0044] FIG. 2 is an illustration of the chemical structures of the VAN and DAP antibiotics.

[0045] FIG. 3 is an illustration of the chemical structure of SEV that illustrates functional groups of SEV. At the physiological pH, ~ 40% of the amine groups are in the form of amine hydrochloride, i.e. , protonated amine, and ~ 60% are free bases, providing ion exchange and hydrogen bonding sites. In SEV chemical structure, a + b = 9, number of primary amines (n) = 0.4, number of protonated amines per monomer (c) = 1, number of crosslinking groups (m) is large, showing a large polymeric molecule.

[0046] FIG. 4 is a graph illustrating equivalent size distribution of SEV particles, measured via analyzing the optical microscopy images. A representative microscopy image of SEV is shown in the inset.

[0047] FIG. 5 is a graph illustrating an SEV-mediated antibiotic R, showing that SEV (4 mg mL’r) removes ~ 95% of VAN or DAP at an initial antibiotic concentration of 1 mg mL-1after 4 h of incubation in conducted evaluation work. The 95% R values are likely to be underestimated as the corresponding VAN Cewas lower than the detection limit of the UV-vis spectrophotometer.

[0048] FIG. 6 is a table illustrating the percentage of protonated amine groups and C / N atomic ratio, estimating the fraction of each compound.

[0049] FIG. 7 is a high resolution N Is scans of SEV, analyzed by curve fitting with respect to the nitrogen (N Is), non-protonated N-C, and protonated N+-C species.

[0050] FIG. 8 is a high resolution N Is scans of VAN, analyzed by curve fitting with respect to the nitrogen (N Is), non-protonated N-C, and protonated N+-C species.

[0051] FIG. 9 is a high resolution N Is scans of DAP, analyzed by curve fitting with respect to the nitrogen (N Is), non-protonated N-C, and protonated N+-C species.

[0052] FIG. 10 is a high resolution N Is scans of SEV-VAN, analyzed by curve fitting with respect to the nitrogen (N Is), non-protonated N-C, and protonated NT-C species.

[0053] FIG. 11 is a high resolution N Is scans of SEV-DAP, analyzed by curve fitting with respect to the nitrogen (N Is), non-protonated N-C, and protonated N~-C species.

[0054] FIG. 12 is ATR-FTIR spectra of VAN, DAP, SEV, SEV-VAN, and SEV-DAP.

[0055] FIG. 13 is a graph illustrating a time-change of SEV (4 mg mL-1)-mediated VAN or DAP (1 mg mL-1) removal percentage (R), showing that > 80% of the antibiotics is removed in ~ 5 min in conducted evaluation work. The data points within the dashed black bracket indicate that the corresponding VAN Cewas lower than the detection limit; thus, the R ~ 95% is likely to be underestimated.

[0056] FIG. 14 is a graph illustrating a time-change of VAN (20 mg mL-1) or DAP (16 mg mL-1) removal capacity (qe), showing that SEV (4 mg mL-1) reaches a maximum removal capacity in ~ 12 h for VAN or ~ 4 h for DAP in conducted evaluation work.

[0057] FIG. 15 is a schematic illustration of a porous spherical SEV particle, discretized to m points in the radial direction, representing the exemplary variables involved in the diffusion- adsorption process of antibiotics. Here, E denotes the porosity.

[0058] FIG. 16 is a graph illustrating a fractional coverage (0) of SEV (4 mg mL-1) with VAN (20 mg mL-1) or DAP (16 mg mL-1) as a function of incubation time, calculated from the experimental data and theoretical model described by Equations (3), (4), and (5) (R2= 0.90 forVAN or 0.98 for DAP), which shows that the SEV adsorption time constant is ~ 55.6 s for VAN or ~ 1.1 s for DAP.

[0059] FIG. 17 is a graph illustrating an effect of initial VAN or DAP concentration (Co) on the maximum removal capacity (qe) of SEV (4 mg mL-1) after 6 h or 12 h of incubation, respectively in conducted experimentation work.

[0060] FIG. 18 is a graph illustrating an effect of pH on the VAN or DAP (1 mg mL-1) removal percentage (R) of SEV (4 mg mL-1) from conducted evaluation work, showing a significant reduction of VAN removal at pH < 6, and a slight reduction of DAP removal at pH < 3.

[0061] FIG. 19 shows a schematic illustration and a table including pKa values of the major functional groups of VAN, DAP, and SEV along with their theoretical positive, negative, and net charges at varying pH.

[0062] FIG. 20 is a graph illustrating VAN or DAP (Co = 1 mg mL-1) removal percentage (R) without SEV at varying pH.

[0063] FIG. 21 is a graph illustrating SEV-mediated VAN or DAP (1 mg mL-1) R at varying concentrations of Na+during conducted evaluation work. The data points within the dashed brackets indicate that the corresponding VAN Ceor DAP Cewas lower than the detection limit, where Ct(mg mL-1) denotes the equilibrium DAP or VAN concentration after removal. Thus, R ~ 95% in all the panels is likely to be underestimated.

[0064] FIG. 22 is a graph illustrating SEV-mediated VAN or DAP (1 mg mL-1) R at varying concentrations of Ca2+during conducted evaluation work. The data points within the dashed brackets indicate that the corresponding VAN Ceor DAP Ce was lower than the detection limit. Thus, R ~ 95% in all the panels is likely to be underestimated.

[0065] FIG. 23 is a graph illustrating SEV-mediated VAN or DAP (1 mg mL-1) R at varying concentrations of bile acid (BA) during conducted evaluation work. The data points within the dashed brackets indicate that the corresponding VAN Ceor DAP Cewas lower than the detection limit. Thus, R ~ 95% in all the panels is likely to be underestimated.

[0066] FIG. 24 is a graph illustrating SEV-mediated VAN or DAP (1 mg mL-1) R at varying concentrations of maleic acid (MA) during conducted evaluation work. The data points within the dashed brackets indicate that the corresponding VAN Ceor DAP Cewas lower than the detection limit. Thus, R ~ 95% in all the panels is likely to be underestimated.

[0067] FIG. 25 is a graph illustrating SEV-mediated VAN or DAP (1 mg mL-1) R at varying concentrations of phosphatidylcholine (PC) during conducted evaluation work. The highlighted regions in panels show the physiological concentration range of each compound in the small intestine. The pH of all samples was adjusted to 6.5. The data points within the dashed brackets indicate that the corresponding VAN Ceor DAP Cewas lower than the detection limit. Thus, R ~ 95% in all the panels is likely to be underestimated.

[0068] FIG. 26 is a graph illustrating VAN or DAP (Co = 1 mg mL-1) R without SEV at varying PC concentrations.

[0069] FIG. 27 is a graph illustrating VAN removal as a percentage (R) as capacity (qe) of SEV (4 mg mL-1) at varying initial VAN concentrations, obtained in the fasted state simulated intestinal fluid (FaSSIF) and fed state simulated intestinal fluid (FeSSIF) during conducted evaluation work.

[0070] FIG. 28 is a graph illustrating VAN removal as capacity (qe) of SEV (4 mg mL'1) at varying initial VAN concentrations, obtained in the FaSSIF and FeSSIF during conducted evaluation work.

[0071] FIG. 29 is a graph illustrating R at varying initial DAP concentrations, obtained in the FaSSIF and FeSSIF during conducted evaluation work.

[0072] FIG. 30 is a graph illustrating varying qeof SEV (4 mg mL-1) at varying initial DAP concentrations, obtained in the FaSSIF and FeSSIF during conducted evaluation work.

[0073] As it concerns the graphs of FIGS. 27-30, the non-significant difference in most of the initial VAN or DAP concentrations shows that food-induced changes in the SIF composition have minor impacts on the antibiotic removal. The pH was maintained at 6.5 in all the experiments. Data are presented as mean ± standard deviation; n = 3. *** p < 0.001, *p < 0.05, and non-significant (N.S.) p ≥ 0.05, representing the statistical differences between the 2 data points of FaSSIF and FeSSIF. The data points within the black dashed bracket indicate that the corresponding VAN Cewas lower than the detection limit. Thus, R ~ 95% is likely to be underestimated in FIGS. 27-30.

[0074] FIG. 31 is a graph illustrating VAN or DAP qeof SEV (4 mg mL-1) versus equilibrium VAN or DAP concentration after 12 h or 6 h of incubation, respectively.

[0075] FIG. 32 is a schematic flow chart illustrating an exemplary process for microdilution, implemented in conducted evaluation work to assess the bioactivity of uncaptured antibiotics after incubation with SEV.

[0076] FIG. 33 is a graph illustrating the bacterial density (ODeoo) of VRE / zw following exposure to the uncaptured DAP by SEV (4 mg mL-1) at incubation times varying from 5 min to 4 h during conducted evaluation work. After 5 min, DAP had no antibiotic activity against VRE / m.

[0077] FIG. 34 is a graph illustrating ODeoo of VSE / m following exposure to the uncaptured VAN by varying doses of SEV after 4 h incubation during conducted evaluation work. The available antibiotic against VSE / m was decreased by increasing the SEV dose, confirming SEV dose-dependent VAN removal.

[0078] FIG. 35 is a graph illustrating the bacterial density (ODeoo) of VSE / m following exposure to the uncaptured VAN (initial concentration = 128 pg ml.'1) by SEV (4 mg mF1) at incubation periods varying form 5 min to 4 h

[0079] FIG. 36 is a graph illustrating the OD(>oo of VSE / wz following exposure to the uncaptured VAN (initial concentration = 128 pg mL-1) by varying doses of SEV after 4 h of incubation.

[0080] FIG. 37 is a graph illustrating ODooo of VSE / zzz following exposure to the uncaptured VAN by SEV (40 mg mL-1) at incubation times varying from 5 min to 4 h during conducted evaluation work. The available antibiotic against VSE / hz decreased by increasing the incubation time, showing time-dependent VAN removal using SEV.

[0081] FIG. 38 is a graph illustrating VAN removal percentage (A) of sevelamer hydrochloride (used for in vitro studies, 4 mg mL-1) and sevelamer carbonate tablets (used for in vivo studies after crushing, 5.5 mg mL-1, considering that each tablet has 73 ± 5% active SEV ingredient) at varying initial VAN concentration (Co) after 12 h of incubation. The data point within the purple region indicates that the corresponding VAN Ceis lower than the detection limit. Thus, R ~ 95% is likely underestimated.

[0082] FIG. 39 is a schematic illustration of an exemplary timeline of a mouse model used to assess the anti-VAN capability of SEV in vivo during conducted evaluation work.

[0083] FIG. 40 is a graph illustrating E.faecium fecal density from conducted evaluation work (E.faecium gene copy number mg-1of feces as assessed by quantitative PCR) with or without SEV treatment.

[0084] FIG. 41 is a graph illustrating Cumulative shedding of total E. faecium, defined as the area under the E. faecium gene copy number curve (AUC) from time of bacterial inoculation (day 0) through 14 days after the inoculation from conducted evaluation work.

[0085] FIG. 42 is a graph illustrating VanA gene fecal density (VanA gene copy number mg-1of feces) as assessed by quantitative PCR with or without SEV treatment during conducted evaluation work.

[0086] FIG. 43 is a graph illustrating cumulative VanA gene shedding, defined as the area under the VanA gene copy number curve (AUC) from time of inoculation (day 0) through 14 days after inoculation.

[0087] FIG. 44 is a graph illustrating the relative proportion of VanA gene density (VanA gene density / E. faecium gene density) through 14 days after the inoculation with or without SEV treatment from conducted evaluation work.

[0088] FIG. 45 is a graph illustrating the AUC proportion of the VanA gene shedding (VanA gene AUC / E.faecium gene AUC) through 14 days after the inoculation.

[0089] Data in FIGS. 42-45 are presented as mean ± standard deviation; n = 5 per group for each experimental condition. For all figures, mice inoculated at a 1:2.5 ratio of VREfm to VSEfm arein blue and mice inoculated at a 1:1000 ratio of VREfm to VSE / m, are in red. * p < 0.05 and “ns” p > 0.05.DETAILED DESCRIPTION OF THE INVENTION

[0090] The following description is of exemplary embodiments presently contemplated for carrying out the present invention. This description is not to be taken in a limiting sense but is made merely for the purpose of describing the general principles and features of the present invention. The scope of the present invention should be determined with reference to the claims.

[0091] FIG. 1 provides an illustration of an exemplary embodiment of a process for the removal of one or more antibiotic agents within a patient’s GI tract while that patient is undergoing one or more intravenous (IV) antibiotic treatments via one or more intravenous injections. The IV treatment can include injection of Vancomycin (VAN) alone, an injection of VAN followed by at least one injection of daptomycin (DAP), or a treatment process that can involve injection of both DAP and VAN into a patient (e.g. at the same time, at different times, etc.) such that both DAP and VAN may be present in the patient’s body for a period of time. In other embodiments, the antibacterial IV injection treatment can include another combination of antibacterial compounds that include VAN alone or VAN in combination with one or more other antibacterial compounds (e.g. linezolid, DAP, etc.). In yet other embodiments, it is contemplated that the antibacterial IV treatment can include one or more antibacterial compounds that involve other antibacterial agents (e.g. linezolid, DAP, etc.).

[0092] The patient can orally take one or more capsules, pills, or other type of orally configured receptacle that can include one or more antibiotic removal agents or one or more antibiotic deactivation agents (e.g. at least one antibiotic removal agent, at least one antibiotic deactivationagent, etc.). The orally taken antibiotic removal agent or antibiotic deactivation agent can be taken as a powder that is swallowed via the mouth of the patient in powder form as after the powder is mixed into a liquid (e.g. water) for the patient to drink. The orally digestible antibiotic removal agent or antibiotic deactivation agent can also be in a pill form, bin a pill form that is coated with a coating, or be in some other suitable form that can be digested directly (e.g. without being coated or positioned within a capsule or other type of oral delivery mechanism) for digestion of the agent(s) by the patient for the agent(s) being delivered to the patient GI tract via its oral digestion. The patient can be animal (e.g. a human, a pet, etc.). The oral digestion of the antibiotic removal agent or antibiotic deactivation agent can occur during the IV injection, before the IV injection, or shortly after the IV injection in different embodiments. The oral digestion of the antibiotic removal agent or antibiotic deactivation agent can alternatively occur during or shortly after the antibiotic is taken by the patient in any other type of antibiotic administration or treatment protocol (e.g. taken orally by the patient as a pill, etc.).

[0093] For example, in some embodiments, the oral digestion of the antibiotic removal agent or antibiotic deactivation agent can be timed to occur so that the antibiotic removal agent or antibiotic deactivation agent can be digested and subsequently be present within the patient GI tract so that after the IV injection of one or more antibacterial agents, the antibiotic removal agent or antibiotic deactivation agent is present in the GI tract for removal or deactivation of any of the antibiotic compounds from the patient GI tract so that any antibiotic compound(s) that may pass into the patient’s GI tract after being injected into the patient can be effectively removed from the GI tract or effectively deactivated from the GI tract (e.g. adsorbed, absorbed,or otherwise biologically inserted via interaction between the antibiotic removal agent or antibiotic deactivation agent and the antibiotic compound(s)).

[0094] In some embodiments, the patient can be on an oral digestion protocol for repeatedly taking the antibiotic removal agent(s) or antibiotic deactivation agent(s) over the course of a period of time (e.g. every 2 hours, every 4-6 hours, every 4-12 hours, etc.) during the time the patient may undergo the IV injections of the antibiotic compound(s). In other embodiments, the oral digestion may be timed to occur before, during or after an antibiotic injection or antibiotic treatment is administered.

[0095] As can be appreciated from the right side of the schematic illustration of FIG. 1, the antibiotic removal agent(s) or antibiotic deactivation agent(s) can be configured to react with any excess antibiotic compound present in the patient GI tract after the patient is injected with the antibiotic compound(s). The removal can occur via the antibacterial compounds being adsorbed into the antibiotic removal agent(s) or antibiotic deactivation agent(s) due to an affinity the antibiotic removal agent(s) can have for the antibiotic compounds. The removal may also occur by a chemical reaction or other type of interaction with the antibiotic removal agent(s) or antibiotic deactivation agent(s) and the antibiotic compound(s) in the GI tract that reduces or eliminates the bioreactivity of the antibiotic compounds so that bacteria within the IG tract are unable to react to the antibiotic compound and are not affected by that compound after it reacts with the antibiotic removal agent(s) or antibiotic deactivation agent(s) in the patient GI tract.

[0096] As can be appreciated from a comparison of the left side of FIG. 1 with the right side of FIG. 1, the use of the antibiotic removal agent(s) or antibiotic deactivation agent(s) can avoid antibiotic compounds being bioreactive in the patient’s GI tract so that those compounds do notkill healthy gut bacteria of the patient and also avoid antibiotic resistance evolution by causing such antibiotic resistant bacteria to be produced in an increasing proportion in the patient’s GI tract while that patient undergoes an IV antibiotic treatment or other type of antibiotic treatment.

[0097] In some embodiments, the agents can be adapted to help protect microbiome within the body of an animal or a human.

[0098] FIG. 15 provides an illustration of an exemplary antibiotic removal agent or antibiotic deactivation agent that is structured in a particulate form that has pores that adsorb and / or absorb the antibiotic compounds within the GI tract, for example. The antibiotic removal agent or antibiotic deactivation agent can render the antibiotic compounds inert to the GI bacteria and can be passed out of the patient via the patient’s feces or urine without having any significant bioreactivity for bacteria due to the functionality of the antibiotic removal agent particulate(s) or the antibiotic deactivation agent particulate(s). The antibiotic removal agent(s) or antibiotic deactivation agent(s) consumed by the patient may then be passed out of the patient via urine or feces so the antibiotic compound(s) are inert (e.g. non-bioreactive) to bacteria as well.

[0099] Embodiments of our process can be performed in a hospital or other type of care facility (e.g. doctor office, urgent care facility, etc.). It is also contemplated that embodiments can be performed in a farm setting to treat farm animals to help prevent the evolution of antimicrobial resistant bacteria in farm animals (e.g. cattle, cows, chickens, pigs, turkeys, poultry, fish, etc.). Embodiments of the process can also be employed to facilitate administering of one or more antibiotic removal agents or one or more antibiotic deactivation agents at different times each day while a patient is undergoing IV injections of one or more antibiotics for treatment of an infection that is not located in the patient’s GI tract.

[0100] As discussed further herein, we have found that some embodiments of our process can be utilized in conjunction with VAN so that VAN can be removed from the GI tract and / or have VAN’s bioreactivity with bacteria removed so that the removed VAN does not affect the patient’s GI tract bacteria (e.g. it can avoid the GI tract bacteria from dying due to the antibacterial nature of the VAN after adsorption and / or absorption by the antibiotic removal agent). Examples of an antibiotic removal agent or antibiotic deactivation agent that can be orally ingested by a patient via the patient’s mouth as an antibacterial agent removal compound or antibacterial agent deactivation compound can include one or more of the following: Veltassa (generic name: patiromer), Kionex (generic name: sodium polystyrene sulfonate), Lokelma (generic name: sodium zirconium cyclosilicate), Kayexalate (generic name: sodium polystyrene sulfonate), Kalexate (generic name: sodium polystyrene sulfonate), Colestipol (sold under the trade names Colestid and Cholestabyl), Tolevamer (sold under trade names: Kayexalate, Kionex, Resonium Calcium, and Solystat), Polacrilex resin (Amberlite IRP64), Polacrilin potassium (Amberlite IRP88), WelChol (colesevelam), sevelamer (SEV), magnesium-containing compounds, calcium-containing compounds, iron-containing compounds, zinc-containing compounds, antacids containing aluminum or magnesium, proton pump inhibitors, or combinations of these materials. For example, one or more of these materials, when ingested by the patient, can function to block the activity of VAN or DAP at physiologically relevant concentrations to effectively remove the antibiotic compounds from the patient’s GI tract so that the patient’s gut bacteria does not react to those compounds (e.g. does not die, does not die in a way that results in driving the uptake of resistant bacteria strains in evolution of the gut bacteria, etc.). This removal can occur via adsorption, diffusion, and / or absorption of the antibacterialcompound(s) in the patient GI tract via the antibiotic removal agent having or being one or more antibacterial agent removal compounds or antibacterial agent deactivation compounds in some embodiments.

[0101] For example, SEV, which is an FDA approved weakly basic anion-exchange polymeric material, can provide antibiotic drug removal from the GI of a patient for DAP as well as VAN. SEV can include polyallylamine that is crosslinked with epichlorohydrin. SEV can be a partial hydrochloride salt that has approximately 40% amine hydrochloride and 60% sevelamer base in some configurations. The amine groups of sevelamer can become partially protonated in the intestine and can interact with phosphate ions through ionic bonding and / or hydrogen bonding.

[0102] As an exemplary embodiment of an antibiotic removal agent or an antibiotic deactivation agent, we performed experimental work to investigate the use of SEV as such an agent to be ingested by a patient for passing into the patient’s GI tract (e.g. via oral digestion or via digestion through a feeding tube, etc.) to evaluate how embodiments of our process can work at treating a patient undergoing an antibacterial IV treatment that uses VAN or uses VAN and DAP to evaluate the efficacy SEV could provide at helping to limit or avoid excess antibiotic compounds within the patient’s GI tract so preserve the patient’s per-existing gut bacteria and help avoid evolution of antibiotic resistance bacteria within the patient’s GI tract.

[0103] For example, as discussed below, we conducted SEV-mediated VAN and DAP removal via conducting in vitro experiments at controlled initial antibiotic concentrations, pH, and ionic strengths. The mechanisms of antibiotic removal by SEV were also examined under the influence of individual intestinal fluid components, e.g., bile acid (BA), maleic acid (MA),phosphatidylcholine (PC), and the whole simulated intestinal fluid (SIF). To identify the transient roles of adsorption and diffusion in the antibiotic removal process, a time-dependent model was also developed during the conducted evaluation work. Furthermore, SEV-mediated antibiotic removal was assessed via a standard antibacterial microdilution assay against Enterococcus f aecium (VSEfm) and V AN -resistant Enterococcus faecium (VREfm), and its efficacy for certain embodiments of our process using SEV was also assessed in mouse models of Enterococcus faecium intestinal colonization. Our conducted evaluation work, which is discussed further below, showed that embodiments of our process can provide a translatable adjuvant therapy to combat (or eliminate) AMR that may be induced by VAN or induced by a combination of VAN and DAP. An Appendix is included herewith, which includes additional information on conducted evaluation work discussed herein as well.EXAMPLESMethods and Materials

[0104] In our conducted evaluation and experimentation work, Vancomycin hydrochloride (VAN, United States Pharmacopeia grade, > 95 wt%) and daptomycin (DAP, > 94 wt%) were purchased from VWR International, USA, and Tokyo Chemical Industry, Japan, respectively. Sevelamer hydrochloride resin (> 99 wt%) was supplied from Chemscene, USA, which was used for conducting all in vitro experiments. Sevelamer carbonate (Renvela, > 73 wt%) tablets were sourced by Safecore Health company, USA, and used for the in vivo studies. NaCl (> 99.5 wt%), CaCh-2H2O (for molecular biology, > 99.0 wt%), sodium hydroxide (NaOH, ACS Reagent, > 97 wt%), hydrochloric acid (HC1, ACS reagent, 37% v / v), phosphatidylcholine (PC, type XVI-E, > 99.0 wt%), and cellulose acetate (CA) centrifuge tubefilters with a pore size of 0.22 pm were purchased from Sigma-Aldrich, USA. Maleic acid(C4H4O4, > 98 wt%) was procured from Beantown Chemical Corporation, USA. Fasted-state simulated intestinal fluid (FaSSIF-V2) powder was supplied from Biorelevant, UK. Sodium taurocholate (C26H44NNaO7S, > 95 wt%), used as a bile acid, was purchased from Spectrum Chemical, USA. In all experiments, ultrapure (Milli-Q) water (resistivity ~ 18.2 mQ cm), produced from the deionized water using an ultrafilter (Biopak Polisher, Millipore, USA), was used. Anhydrous ethanol (200 proof) was obtained from KOPTEC, USA.

[0105] Cation-adjusted Mueller Hinton II Broth (CAMHB, BD Difco, USA) was purchased from Becton, USA. E. f aecium strains, namely HD D8, BL00239-1, and BL00239-1R, were derived from a sample isolated from a patient with a VRE / m blood stream infection while being treated at the University of Michigan hospital. Phosphate-buffered saline (PBS) solution (IX) was supplied from Thermo Fisher Scientific (USA). Glycerol (Molecular biology grade) and 1.5 mL microcentrifuge tubes were obtained from VWR International (Molecular biology grade, USA). Swiss-Webster mice were provided by Charles River (USA). Enterococcosel Agar was purchased from BD Difco (BBL formulation, USA). The 5053-PicoLab Rodent Diet 20 was supplied from LabDiet (USA). Ampicillin and veterinary grade VAN for mouse injection were purchased from Alfa-Aesar and Slate Run Pharmaceuticals (USA), respectively. Brain Heart Infusion (BHI) broth was supplied from BD Difco (BBL formulation, USA). Flat natural toothpicks were purchased from Dixon Ticonderoga Company (USA).

[0106] MagMAx 96 DNA Multi-Sample Kit, including DNA binding beads, Multi- Sample DNA Lysis Buffer, and DNA Elution Buffer, were purchased from Thermo Fisher Scientific (USA). Tris(hydroxyethyl)aminomethane hydrochloride (Tris-HCl, > 99.0 wt%),sodium ethylenediaminetetraacetic acid (EDTA, 2 mM), Triton X-100, isopropanol (99% v / v), lysozyme (> 23500 units mg-1protein), and 96 deep well plates were purchased from VWR (USA). Mutanolysin (> 4000 units mg-1protein) and bovine serum albumin (BSA, > 98.0 wt%) were purchased from Millipore-Sigma (USA). “PerfeCta qPCR FastMix Low Rox” qPCR supermix reagent was obtained from Quantabio (USA). E. faecium superoxide dismutase (sodA) gene specific primers and probe were designed using Primer Express from Applied Biosystem (USA). VanA ligase specific primers and probe were designed using Primer Quest from Integrated DNA Technologies (IDT, USA).

[0107] SEV Particle Size Measurement: To determine the particle size, microscopy images of SEV (10 mg) dispersed in the ultrapure water (1 mL) were acquired using an inverted microscope (Nikon ECLIPSE TE300, Japan) with a 10X objective lens and a total magnification of 100X. The area of ~ 500 individual particles was measured using ImageJ software (version 1.53124), which was used to calculate the equivalent diameter of each particle, assuming that the particles were spherical.

[0108] SEV-mediated DAP and VAN Removal: The antibiotic removal experiments were carried out in a batch process. DAP or VAN stock solutions were independently prepared by dissolving 200 mg of VAN in 5 mL of ultrapure water (final concentration = 40 mg mL-1) or 100 mg of DAP in 5 mL of ultrapure water (final concentration = 20 mg mL-1). Then, VAN or DAP solutions with varying concentrations (1-24 mg mL-1of VAN or 1-16 mg mL-1of DAP) were prepared by the consecutive dilution of stock solutions with ultrapure water. The solution pH was adjusted to 6.5 via adding a 0.5 M NaOH solution. Desirable amounts of SEV were added to the VAN or DAP solutions, vortexed for 5 min, and placed on a nutating mixer(Fisherbrand, USA) to gently agitate at 60 rpm for a given incubation period. Afterwards, the mixtures were centrifuged (5000 Xg, 5 min), and the supernatants were separated and analyzed using a UV-vis spectrophotometer (Tecan Model Infinite 200 Pro, USA) at the wavelength of maximum absorbance (Xmax), i.e., 280 nm for VAN or 364 nm for DAP, to determine the unadsorbed (free) antibiotic concentration.

[0109] UV-vis Spectroscopy for DAP and VAN Concentration Measurements: To measure DAP or VAN concentration, calibration lines were obtained for all experimental conditions, including 12 lines for VAN and 13 lines for DAP via recording the absorbance of VAN (Xmax= 280 nm) or DAP (A.max= 364 nm) solutions at known antibiotic concentrations using the UV-vis spectrophotometer. The calibration lines were used to calculate the unadsorbed (free) antibiotic concentration in the supernatant after contact with SEV.

[0110] SEV-Mediated DAP and VAN Removal Kinetics: To investigate the antibiotic adsorption kinetics, DAP or VAN solutions at low (1 mg mU1) or high (16 mg mU1of DAP or 20 mg mL-1of VAN) concentrations were prepared. The final volume of antibiotic solutions was 1 mL after adjusting the pH to 6.5 using a 0.5 M NaOH solution. Then, SEV (4 mg) was added to the antibiotic solutions, vortexed for 5 min, and placed on a nutating mixer to gently agitate (60 rpm, up to 24 h). At varying time intervals, the mixtures were centrifuged (5000 Xg, 5 min), and the supernatants were collected to assess using the UV-vis spectrophotometer at the Xmaxof DAP (364 nm) or VAN (280 nm) to determine DAP or VAN concentration, respectively. The antibiotic removal capacity of SEV at time t was calculated using Equation (1):

[0111] where q (mg g"1) is the equilibrium amount (mg) of antibiotic (VAN or DAP) adsorbed per 1 g of SEV at time t, Co (mg mL-1) represents the initial antibiotic concentration, C (mg mL"1) is antibiotic concentration in the supernatant at time t, m (g) denotes SEV mass, and V (mL) is the volume of antibiotic solution.

[0112] Equilibrium batch adsorption measurements were conducted similarly after 4 h of SEV incubation with the antibiotic solution to determine the maximum DAP or VAN removal capacity of SEV. Removal percentage (R, %) and equilibrium removal capacity (qe, mg mL-1) were obtained using Equations (2 )and (3), respectively:

[0113] where Ce(mg mL-1) denotes the equilibrium DAP or VAN concentration after removal. Note that the lower limit of detection for VAN or DAP concentration by UV-vis spectrophotometry was ~ 0.05 mg mL-1. When the measured VAN or DAP Cewas below the detection limit, it was assumed to be 0.05 mg mL-1. Therefore, any R value ~ 95% calculated using this assumption is likely underestimated.

[0114] Effect of pH on the SEV-Mediated DAP or VAN Removal: The SEV-mediated removal of VAN (1 mg mL-1) or DAP (1 mg mL-1) at pH — 1.5 - 10 was studied to assess the effect of functional group ionizations on SEV-antibiotic interactions. The pH of antibiotic solutions was modified via adding 0.5 M HC1 or 0.5 M NaOH solutions to the stock antibiotic solutions. Then, 4 mg of SEV was added to the VAN or DAP solutions at varying pH, vortexed for 5 min, and placed on a nutating mixer at 60 rpm for 4 h. The unadsorbed DAP or VANconcentration (Ce) was measured via collecting the supernatants after centrifugation (5000 Kg, 5 min) and recording the absorbance using the UV-vis spectrophotometer at Xmax= 364 nm or Xmax= 280 nm, respectively. The R and qcwere calculated using Equations (2) and (3), respectively.

[0115] Effects of Ion Type and Ionic Strength on the SEV-Mediated DAP Or VANRemoval: To examine the effect of monovalent or divalent ions on the SEV-DAP or SEV-VAN interactions, the SEV-mediated removal of DAP (1 mg mL-1) or VAN (1 mg mL-1) at varying sodium chloride (NaCl) or calcium chloride (CaCh) concentrations (i.e., 10 mM, 50 mM, 100 mM, or 200 mM) was conducted. The pH of DAP or VAN solutions was maintained at 6.5 using a NaOH solution (0.5 M). Then, SEV (4 mg) was added to the antibiotic solutions including varying concentrations of sodium (Na+) or calcium (Ca2+) ions. The SEV-VAN or SEV-DAP solutions were vortcxcd (5 min) and placed on a nutating mixer (60 rpm, 4 h). The DAP or VAN concentration in the supernatant (Ce) was measured via collecting the supernatant after centrifugation (5000 *g, 5 min) and recording the absorbance using the UV-vis spectrophotometer at Xmax=364 nm or Xmax=280 nm, respectively. The R and qewere obtained using Equations (2) and (3), respectively.

[0116] Effects of SIF Components on the SEV-Mediated DAP or VAN Removal: The SEV- mediated removal of DAP (1 mg mL-1) or VAN (1 mg mL-1) at varying concentrations of BA (0- 20 mM), MA (0-100 mM), or PC (0-5 mM) was conducted to investigate the effects of SIF components on SEV-DAP or SEV-VAN interactions. For all tests, the pH of DAP or VAN solutions was maintained at 6.5 using a 0.5 M NaOH solution, and the total sample volume was 1 mL. SEV (4 mg) was mixed with the DAP or VAN solutions, containing an SIF component at varying concentrations. The mixtures were then vortexed (5 min) and placed on a nutating mixer(60 rpm, 4 h). The unadsorbed DAP or VAN concentration (Ce) was measured via collecting the supernatants after 5 min of centrifugation at 5000 Xg and recording the absorbance using the UV-vis spectrophotometer at Xmax= 364 nm and Xmax= 280 nm, respectively. The R and qcwere obtained using Equations (2) and (3), respectively.

[0117] SIF Preparation: To emulate the small intestinal fluid in vitro, fasted-state SIF and fed- state SIF were prepared in accordance with standard protocols. To prepare the fasted-state SIF solution, NaOH pellets (1.392 g), maleic acid (2.22 g), and NaCl (4.01 g) were dissolved in ultrapure water (0.99 L). The final volume of the solution was maintained at 1 L using ultrapure water after adjusting the final pH to 6.5 using a 0.5 M NaOH solution. Then, fasted-state SIF-V2 powder (1.79 g) was added and stirred for 1 h at ambient temperature. To prepare the fed-state SIF solution, sodium taurocholate (8.25 g) and PC (2.95 g) were dissolved in the fasted-state SIF solution (total volume = 1 L). After addition, the solution was stirred for 4 h at ambient temperature.

[0118] Effect of SIF on the SEV-Mediated Removal of DAP or VAN: To examine the competitive effect of SIF on the antibiotic removal capability of SEV, antibiotic solutions with concentrations in the range of 1 mg mL-1to 20 mg mL-1for VAN or 1 mg mL-1to 16 mg mL-1for DAP were prepared via diluting the corresponding antibiotic stock solutions with fasted-state SIF or fed-state SIF. The final volume of antibiotic solutions was 1 mL after adjusting the pH to 6.5 using a 0.5 M NaOH solution. SEV (4 mg) was then added to the DAP or VAN solutions, vortexed for 1 min, and placed on a nutating mixer at 60 rpm. After 4 h of incubation, samples were centrifuged (5000 Xg, 5 min), and the supernatants of DAP or VAN solutions wereseparated and assessed using the UV-vis spectrophotometer at Xmax= 364 nm and Xmax= 280 nm, respectively. The 7? and qewere calculated using Equations (2) and (3), respectively.

[0119] Attenuated Total Reflectance (ATR)-Fourier Transform Infrared (FTIR)Spectroscopy: To identify the interactions between SEV and antibiotics as well as the functional groups of SEV, VAN, and DAP, the infrared spectra of freeze-dried samples were acquired using a Vertex 70 spectrometer (Bruker Optics) in accordance with the Bouguer-Beer-Lambert law. For this purpose, the samples were frozen at — 80 °C and lyophilized at pressure ~ 0.01 mbar for 48 h before measurements. The spectrometer had a liquid nitrogen cooled mercury-cadmium- telluride (MCT) detector. Measurements were conducted in the ATR geometry using a Dimax (Harrick scientific, USA) diamond ATR accessory. For each spectrum, a total of 500 scans were averaged with a 6 cm"1resolution in wavenumbers ranging from 500 cm"1-4000 cm"1. The absorbance was calculated by referencing to the spectrum of clean diamond crystal.

[0120] X-ray Photoelectron Spectroscopy (XPS): To investigate the interactions between SEV and the antibiotics, XPS measurements were conducted with a VersaProbe III instrument (Physical Electronics, Germany), furnished with a concentric hemispherical analyzer (CHA) and a monochromatic aluminum (Al) ka X-ray source with a photon energy of 1,486.6 eV. XPS spectroscopy was performed according to the international organization for standardization, ISO 15472:2001. To neutralize the surface charges, low energy electrons (less than 5 eV) and argon ions were used. The axis of binding energy was calibrated using foils made of copper (Cu) (Cu 2p3 / 2 = 932.62 eV and Cu 3ps / 2 = 75.1 eV) and gold (Au) (Au 4f? / 2 = 83.96 eV) that were sputter cleaned. Measurements were implemented at a takeoff angle of 45° with respect to the sample surface plane, which led to an average sampling depth of 3-6 nm. For homogeneous samples, thestandard deviation of major elements (i.e., > 5 atom%) is less than 3%, whereas minor elements undergo greater variability. The diameter of X-ray beam was about 200 gm. The relative sensitivity factors (RSFs) were used for quantifying the XPS peaks.

[0121] Assessing the Antibiotic Activity of Uncaptured DAP or VAN Using BrothMicrodilution Assay: Broth microdilution assays were conducted to quantify the effects of SEV on the antibiotic activity of DAP or VAN against patient-derived E.faecium. Two previously characterized strains, VAN-susceptible HD D8 (VSE / m) with a VAN minimum inhibitory concentration (MIC) of ~ 1 pg mL-1and DAP-susceptible BL00239-1 (VRE / m) with a DAP- MIC of ~ 2.1 pg mL-1were cultivated for VAN or DAP assays, respectively. SEV-mediated antibiotic removal was conducted in ultrapure water at pH = 6.5, following centrifugation at 16,300 Xg for 5 min. The supernatants were collected and passed through a CA filter with a pore size of 0.22 pm. All assays were established according to the guidelines of the Clinical & Laboratory Standards Institute (CLSI). Initial VAN or DAP concentrations, i.e., the antibiotic concentration prior to SEV-mediated removal, were used to create 2fold serial DAP or VAN dilutions. After the incubation of plates at 35 °C for 24 h, bacterial cell density was recorded using a BioTek Synergy Hl Plate reader (Agilent Technology, USA) at an optical density of 600 nm. The optical density data were fitted to the Hill function as outlined previously, and the reductions in supernatant VAN or DAP concentrations were indicated by right shifts in the fitted growth curves.

[0122] Evaluating SEV Anti-VAN Efficacy In Vivo: A mouse model of intestinal E. faecium colonization was used to assess the in vivo SEV-mediated sequestration of VAN. Two E.faecium strains, VAN-resistant BL00239-1 (VRE / m) and VAN-sensitive HD D8 (VSE / m), were againused with HD D8 being derived from BL00239-1 following mouse GI passage. PCR was used to confirm the loss of VanA expression with HD D8 as described below. All animal protocols were approved by The Pennsylvania State University Institutional Animal Care and Use Committer (PSU-IACUC, Protocol #47581). Given the absence of evidence supporting sex-differences in the biliary secretion of VAN, only female mice were used to minimize research animal use in accordance with PSU-IACUC guidelines. Sample sizes were based on previous experience with the use of this mouse model. Adult female (8-week-old) Swiss Webster mice were subsequently housed under BSL-2 housing conditions for a minimum of 2 weeks at 5 mice per microisolator cage until E.faecium inoculation and singularly thereafter. Mice were given sterile water and irradiated feed (LabDiet, PicoLab Rodent Diet 20 5053) ad libitum for the duration of study. All surfaces were thoroughly cleaned with 70% v / v ethanol, and outer gloves were changed between cages to prevent cross contamination when handling mice.

[0123] To aid in intestinal E.faecium colonization, mice were treated with ampicillin (ad libitum, 0.5 g L- 1in drinking water) for 7 days prior to inoculation. Bacterial isolates were cultured overnight in the BHI broth at 35 °C, and mice were subsequently inoculated via oral gavage with sterile saline (100 μL), containing 108colony forming units (CFUs) of E.faecium at a 1 : 1000 or a 1 : 2.5 ratio of VRE / / ?? (BL00239-1) to VSE / m (HD-D8) for the assessment of SEV-mediated removal of VAN. Mice were then treated twice daily with either 100 μL sterile water or 100 μL sterile water, containing 41 mg sevelamer carbonate (equal to 30 mg of active SEV), for 8 days. Sevelamer carbonate tablets (Renvela) were crushed into a fine powder for use. Given the thick gel-like nature of SEV when added to sterile water, traditional oral gavage was not feasible. Instead, SEV treatment was achieved via directly pipetting the mixture into the backof the mouth, encouraging mice to swallow the mixture voluntarily. Starting one day post inoculation, mice received 60 mg kg-1of VAN, diluted in saline or saline only via subcutaneous injection to assess the capability of SEV in removing VAN in vivo. Both VAN and saline control were administered once daily for a duration of 5 days. At designated timepoints, fresh fecal samples were collected by placing mice in clean unsealed plastic containers until defecation. The resulting fecal pellets were transferred to pre-weighed 1.5 mL microcentrifuge tubes via autoclaved sterile toothpicks, and samples were suspended in PBS (25 μL of PBS per mg of feces) prior to freezing at -80 °C in 25% glycerol for later analysis.

[0124] PCR Quantification of Fecal E. faecium and VanA Density: Sample DNA was extracted on a MagMAX Express 96 Platform (Applied Biosystems, USA) using the MagMAX 96 DNA Multi-Sample Kit) and the “4412021 DW blood” standard pre-loaded extraction protocol. Briefly, 50 μL of frozen fecal samples were incubated with 150 μL of enzymatic lysis buffer consisting of 20 niM Tris-HCl, 1.2% Triton X-100, 2 mM sodium EDTA, 250 U mL-1mutanolysin, and 20 mg mL-1lysozyme, in a 96 deep well plate for 30 min at 37 °C using an Incubating Microplate Shaker heated shaker (VWR, USA) at 1,000 rpm. Post incubation, 100 μL of Multi-Sample DNA lysis buffer was added, followed by 250 μL of isopropanol, and the samples were mixed for 3 min at 1,300 rpm using a microplate shaker (Eppendorf MixMate, USA). DNA binding beads (20 μL) were then added, and the solution was again mixed for 3 min at 1,300 rpm. Samples were run on the MagMAX Express 96 platform using the 4412021 DW blood protocol and eluted in a final volume of 150 μL of DNA Elution Buffer provided in theMagMax 96 DNA Multi-Sample Kit.

[0125] Two sets of PCR primers and probes were designed to quantify fecal concentrations of total E.faecium and the VanA resistance gene. Primers and probes targeting the E.faecium superoxide dismutase (sodA) gene (accession# NC_017960.1) and E. faecium VanA ligase (accession# CAA40215.1) were designed using Primer Express and PrimerQuest, respectively, as shown in Table 1. Real-time quantitative PCR assays were carried out using a 7500 Fast Real- Time PCR System (Applied Bio-systems, USA) with an initial denaturation (95° C, 30 s), followed by 40 cycles of denaturation (95° C, 3 s) and annealing / extension (60° C, 30 s). Each reaction contained 2 μL of extracted DNA in a total volume of 20 μL with the final concentrations of the following components: IX PerfeCta qPCR FastMix Low Rox; 1 pg μL"1BSA; E.faecium primers and probe (750 nM and 50 nM, respectively) or VanA primers and probe (300 nM and 200 nM, respectively). Resulting cycle threshold (Ct) values were interpolated with a standard curve of known concentrations of plasmid derived gene copies to determine sample concentrations per mg of feces.Table 1: Primers and probes used in conducted evaluation and experimentation work

[0126] The statistical analyses were conducted using GraphPad Prism software (version 10.0.3), and mean differences were considered significant when p values < 0.05. Student’s t-test was performed to distinguish any significant difference between two independent groups. For in vivo experiments, discrete measures were analyzed via the Mann- Whitney U test, and repeated measures were analyzed by the mixed-effect analysis via Tukey’s post-hoc test for multiple comparisons.Results

[0127] SEV-Mediated DAP or VAN Removal: FIG. 2 shows the chemical structures of antibiotics VAN and DAP, respectively. Glycopeptide VAN comprises a plethora of ionizable functional groups that are responsive to pH. Accordingly, the net charge of VAN is positive at the physiological pH because of amine group protonation (pKa= 7.75 and 8.89). VAN also has a carboxyl group with pKa= 2.18, inducing local negative charges at the physiological pH. The amine and carbonyl groups also contribute to the specific interactions of VAN with D-Ala-D-Ala in the bacteria cell wall peptidoglycan via forming hydrogen bonds. Lipopeptide DAP bears four pH-dependent carboxyl groups with pKa < 4.5, rendering it negatively charged at the physiological pH, as well as two ionizable amine groups with pKa = 1.3 and 10.7. DAP also has a hydrophobic tail, which is responsible for calcium-dependent bacterial cell membrane disruption. The Ca2+-mediated aggregation of carboxyl-bearing hydrophilic heads induces conformational changes, facilitating DAP interaction with the cell membrane. The hydrophobic part of DAP leads to the formation of sphere-like self-assembled particles at DAP concentrationsexceeding critical micelle concentration (CMC ~ 0.147 mg mL-1), whereas VAN remains as individual molecules.

[0128] FIG. 3 shows the chemical structure of SEV. Anion-exchange SEV polymers in the hydrochloride or carbonate form, comprise crosslinked allyl amines with the amine groups separated by one carbon from the polymer backbone. SEV hydrochloride is hydrophile and swells 6 to 8 times its weight while remaining insoluble in water. The SEV particle equivalent diameter = 102 ± 36 pm, determined via analyzing the optical microscopy images (as shown in FIG. 4). The large particle size prevents the SEV from being absorbed in the G1 tract.

[0129] To examine SEV interactions with VAN or DAP quantitatively, antibiotic removal experiments are conducted via incubating SEV in the antibiotic solutions, prepared using ultrapure water. FIG. 5 presents the VAN or DAP removal percentage (R) of SEV, showing that SEV (4 mg mL-1) removes ~ 95% of the antibiotics (1 mg mL-1) after 4 h, regardless of their electrical charge.

[0130] FIGS. 6-11 present the XPS analyses of SEV, VAN, DAP, SEV-VAN, and SEV-DAP aggregates. The percentage of protonated nitrogen (N+-C) in these materials are shown in FIG. 6, and the corresponding high-resolution N 1 s curves are presented in FIGS. 7-11. The XPS N 1 s peaks are fitted to their constituent subpeaks at ~ 399.5 and ~ 401.3, which are the characteristic C-N species peaks in non-protonated (N-C) and protonated (N+-C) forms, respectively. The N+- C binding energy is 401.9 eV for VAN and 400.9 eV for SEV, which shifts to 401.2 eV in SEV- VAN aggregates. For DAP, the N+-C binding energy is 401.7 eV (SEV N+-C binding energy = 400.9 eV) and shifts to 401.3 eV in SEV-DAP aggregates. Additionally, the weighted average of N+-C percentage for SEV and VAN mixture is ~ 34%, experimentally obtained as ~ 41% forSEV-VAN aggregates. The shift in binding energies and - 7% increase in average N+-C percentage may be a result of VAN interactions with SEV (e.g., hydrogen bonding) despite bearing similar electrical charges, resulting in SEV-mediated VAN removal. The N+-C percentage for the SEV-DAP aggregate (~ 33%) matches the weighted average N+-C percentage of SEV and DAP, i.e., - 31%. Together, the XPS results suggest that SEV has molecular interactions with DAP and VAN.

[0131] The ATR-FTIR spectra of SEV, VAN, DAP, SEV-VAN, and SEV-DAP are presented in FIG. 12. VAN had characteristic peaks at 3276 cm-1, pertaining to the O-H stretching, 1635 cm-1for the C=O stretching in the peptide backbone, 1483 cm-1for the C=C stretching in the aromatic rings, and 1219 cm-1for the phenolic hydroxyl groups. DAP had characteristic peaks at 3276 cm-1for the carboxylic O-H, 2908 cm-1and 2854 cm-1for the C-H stretching, 1627 cm-1for the C=O stretching, 1530 cm-1for the C=C in the aromatic rings, and 1224 cm-1for the C-0 stretching vibrations. SEV had characteristic peaks at 3340 cm-1for N-H stretching, 2901 cm-1and 2856 cm for the C-H stretching, 1563 cm for the N-H bending, and 1313 cm for C-N stretching. In the spectrum of SEV-VAN aggregate, the characteristic peaks of VAN are dominant, and the peaks undergo a minimal shift, possibly because of the limited impact of hydrogen bonding-mediated adsorption on the peak position. The spectrum of SEV-DAP aggregate has a shift in the carboxyl peak of DAP (i.e., from 1627 cm'1to 1646 cm"1), which is likely due to the electrostatic interactions between SEV and DAP.

[0132] Kinetics and Theoretical Considerations of SEV-Mediated DAP or VAN Removal: To examine the time scale of VAN or DAP removal at low (1 mg mL-1) or high (20 mg mL-1for VAN or 16 mg mL-1for DAP) initial antibiotic concentrations, R and removal capacity (<?e) ofSEV at varying incubation times are investigated, as shown in FIGS. 13-16. FIG. 13presents R (SEV concentration = 4 mg ml'1) versus incubation time for VAN or DAP at an initial antibiotic concentration of 1 mg mF1. After 5 min of incubation, R > 80%, which reaches ~ 95% in ~ 3 h, implying the time-independent adsorption of antibiotics. Despite the different chemical structures and charges of antibiotics, VAN and DAP are removed by SEV within a comparable time scale at the experimented SEV and low antibiotic concentrations.

[0133] FIG. 14 shows the time-dependent VAN or DAP qeof SEV at an initial antibiotic concentration of 20 mg mL-1or 16 mg mF1, respectively. The SEV qeincreases for both antibiotics by increasing the incubation time, indicating a transient adsorption process, which reaches an equilibrium state at a plateau qeof 2572 ± 17 mg of VAN per gram of SEV after ~ 12 h and 2857 ± 7 mg of DAP per gram of SEV after ~ 4 h, confirming that DAP is removed faster than VAN, even at a lower initial antibiotic concentration. We hypothesize that the time- regulated adsorption of antibiotics to SEV is associated with molecular diffusion within the SEV pores.

[0134] To test the hypothesis of antibiotic molecular diffusion in SEV, the SEV-mediated removal of VAN or DAP is mathematically modeled. Given that SEV is a highly swellable, porous polymeric resin, the removal process of VAN or DAP molecules is modeled according to dynamic diffusion-adsorption phenomena. SEV is assumed to be a porous sphere with no tortuosity. Moreover, the mass transfer resistance of VAN or DAP from the corresponding bulk solution to the external surface of SEV is neglected as the antibiotic solution is well mixed.Therefore, the bulk VAN or DAP concentration (C) is at a time- and radial (r) position-regulatedequilibrium within the SEV. Taking all these considerations into account, an unsteady-state diffusion-adsorption mass balance is used based on Equation (4) to model the system:

[0135] where the time variations of antibiotic bulk and adsorbed concentrations are balanced by the diffusion of antibiotics (VAN or DAP) in a sphere. In Equation (4), E is SEV porosity(0.167), p is the SEV density (1280 kg m’3, the value reported for polymer resins), q is the VAN or DAP removal capacity at a particular time t, and D is the VAN (7.94 x 10’12nr s-1) or DAP (1.96 x IO’10m2s-1) bulk diffusion coefficient. To solve Equation (4), fractional coverage (9) is considered as q at a given time t divided by the equilibrium (maximum) removal capacity (< / <-). The initial condition and boundary conditions are as follows:

[0136] Initial condition: at t = 0 and r = R, the antibiotic concentration on the SEV surface is equal to the bulk concentration of antibiotic at the beginning of adsorption process (C = Co);

[0137] Boundary condition 1 : time variation of antibiotic bulk concentration is equal to the antibiotic diffusion from the SEV surface to the center

[0138] Boundary condition 2: the SEV center is symmetric, i.e., C( / v?) = C(m-\ ).

[0139] Here, R represents the equivalent radius of SEV particle (51 ± 18 pm), and m represents the number of discretized points along the r direction, as schematically shown in FIG. 15.Equations (5) and (6) describe the relationship of 0 with the adsorption rate constant (kads) and desorption rate constant (kdes).

[0140] where,mads is the SEV mass, no is the initial antibiotic mass per unit mass of SEV normalized with qe, and V is the solution volume. By applying the initial and boundary conditions, Equations (4), (5), and (6) are converted into m sets of ordinary differential equations (ODEs) and numerically solved in Matlab (version R2021 a) using central finite difference for spatial derivatives (method of lines, MOL).

[0141] FIG. 16 presents the time variation of SEV fractional coverage obtained via fitting the experimental data with m = 38,000 and tuning kads. The fractional coverage plot as a function of incubation time with the best fit (R2= 0.90 for VAN or 0.98 for DAP) yielded kads= 0.018 s'1for VAN or kads= 0.9 s"1for DAP, leading to an adsorption time constant (l / kads) of 55.6 s or 1.1 s, respectively. Accordingly, the adsorption of DAP to SEV is more rapid. The small time constant for DAP supports the rapid process of electrostatically driven DAP binding to SEV.Compared with DAP, the time constant of VAN suggests a slower adsorption process, likely as a result of the hydrogen bonding and / or cation-yr interactions. The time (4 h) required to obtain the maximum DAP removal capacity may be ascribed to a diffusion-controlled processes. DAP diffusion within the SEV may be a result of contact-induced deformation and disassembly of otherwise self-assembled DAP molecules. The time required to obtain the maximum VAN removal capacity is longer (12 h), implying that the VAN removal is also a diffusion-controlled process.

[0142] Effect of VAN or DAP Initial Concentration on the Equilibrium Removal Capacity of SEV: The effect of initial VAN or DAP concentration on the q,- of SEV is investigated via incubating SEV in VAN solutions of 1-24 mg mL-1or DAP solutions of 1-16 mg mL-1for 12 or 6 h, respectively. FIG. 17 presents the VAN or DAP qeof SEV at varying initial antibioticconcentrations. As the initial VAN concentration is increased from 1 mg mL-1to 20 mg mL-1, the VAN q.. increases and reaches a plateau of 2570 ± 22 mg g"1. The maximum VAN removal capacity is ~ 17% lower than the theoretical value calculated based on the hydrogen bonding stoichiometric ratio, i.e., 2 mol amine groups of SEV binds to 2 mol (out of 5 mol) of VAN, corresponding to 3116 mg of VAN per gram of SEV. One mol of SEV contains 1.2 mol of protonated amine (NHL) and 1.8 mol of amine (NH2) groups. The slightly lower experimental VAN removal capacity than the theoretical value may be attributed to the inaccessibility of some functional groups due to the electrostatic repulsion between cationic SEV and VAN.

[0143] As the initial DAP concentration is increased from 1 mg mL-1to 12 mg mL-1, the DAP qeof SEV increases and reaches a plateau of 2857 ± 26 mg g-1. The maximum DAP q.- is approximately 120% higher than the theoretical value calculated based on the charge stoichiometry, i.e., 3 mol of protonated amine groups on SEV binds to 3 mol of carboxyl groups on DAP (corresponding to ~ 0.75 mmol or ~ 1215 mg DAP per gram of SEV). The suprastoichiometric DAP removal is likely a result of DAP self-assembly and / or supported lipid bilayer (SLB) formation.

[0144] Effect of pH, Ion Type, Ionic Strength, and SIF Components on the SEV-Mediated DAP or VAN Removal: To investigate the contributions of hydrogen bonding and electrostatic interactions to the SEV-mediated adsorption of VAN or DAP, pH and ionic strength are systematically altered. FIG. 18 shows SEV R for VAN or DAP at varying pH. The orange line shows the intestine pH (~ 6.5). The VAN removal of SEV is ~ 95% over the pH range of 6.5 - 11; however, at acidic conditions, it decreases to ~ 60% at pH = 3 and to ~ 10% at pH = 1.5, possibly as a result of the disruption of hydrogen bonding in the acidic conditions as well as theincreased net positive charge of VAN, increasing the electrostatic repulsion with the positively charged SEV. FIG. 19 presents the chemical structures of VAN, DAP, and SEV, as well as the pKavalues of their major functional groups and corresponding net charges at varying pH. The net charge of VAN and SEV is positive at pH ~ 1.5 to 11, except at pH = 11 wherein VAN is anionic. Therefore, the SEV-mediated VAN removal is likely regulated by hydrogen bonding and / or cation-n interactions with a minimal contribution of electrostatic interactions. The interaction of cationic amine groups of SEV and aromatic rings of VAN may drive cation-7i stacking.

[0145] The SEV qefor DAP remains ~ 95% over a pH range of 3 to 11 (FIG. 18), wherein the net charge of SEV and DAP is positive and negative, respectively. Reducing the pH from 3 to 1.5 decreases the qefor DAP by ~ 10%. At pH < 3, the carboxyl groups of DAP are partially protonated (net charge ~ 0.76), lowering the number of anionic binding sites in DAP that interact with the cationic SEV. The pH-dependent DAP-SEV interactions confirm that the adsorption of DAP to SEV is partially governed by electrostatic attractions. However, other interactions such as hydrogen bonding as well as DAP self-assembly may be involved according to the suprastoichiometric removal capacity. R ~ 90% for DAP at pH 1.5 may be explained based on the following reasons: (i) although DAP has a net positive charge at pH 1.5, some local negative charges still exist on it (as shown in FIG. 19), enabling the electrostatic interactions with the SEV amine groups; (ii) the equilibrium adsorption reactions (rxn.) for SEV-DAP are as follows:

[0146] According to the Le Chatelier’s principle, when the pH is low (e.g., 1.5), the high proton concentration shifts rxn. 2 to the left while the high concentration of protonated amines of SEV shifts rxn. 1 to the right, decreasing the concentration of COO", which subsequently shifts rxn. 2 to the right. Accordingly, the deprotonated carboxyl group concentration of DAP may exceed the theoretical calculation; (iii) DAP may undergo aggregation and phase separation at highly acidic conditions (pH ~ 1.5). The values of R for antibiotics without SEV at varying pH are presented in FIG. 20, indicating that DAP partially precipitates at pH < 2, whereas VAN remains as a stable molecule in the solution and does not precipitate.

[0147] The R and qefor VAN or DAP in electrolyte solutions, comprising either mono- or divalent ions are studied. FIG. 21 shows the SEV (4 mg mL-1) R for VAN or DAP at varying sodium ion (Na+) concentrations. The region highlighted in blue shows the physiological concentration of Na+(~ 155 mM). The R remains almost constant, around 95%, for both antibiotics over the Na+concentration range of 0 - 200 mM. The addition of Na+may reduce the thickness of the electrical double layer without compromising the VAN or DAP surface charge. FIG. 22 shows the SEV-mediated VAN or DAP removal at Ca2+concentrations varying from 0 mM to 200 mM. The region highlighted in red shows the physiological concentration range of Ca2+(~ 2-6 mM). By increasing the Ca2+concentration from 0 mM to 200 mM, the R decreases ~ 10% for VAN and ~ 35% for DAP. The lower R for DAP is a result of carboxyl group neutralization by the divalent cations (Ca2+: COO' = 1 mol : 2 mol). The less pronounced effect of ionic strength on the VAN removal may be a result of non-electrostatic (e.g., hydrogen bonding) driven VAN-SEV binding. The hydrogen bonds are less affected by the ionic strengththan electrostatic bonds. We have observed that when Ca_+concentration is 200 mM, no VAN removal occurs in the absence of SEV; however, the R for DAP is about 10%.

[0148] Given that the intestinal fluid includes lipids and bile salts, which may interact with SEV or the antibiotics, the effect of these components on VAN or DAP removal is investigated in vitro. FIG. 23 shows the SEV-mediated VAN or DAP removal at bile acid concentrations varying from 0 mM to 15 mM, covering the physiological concentration range from fasted-state SIF to fed-state SIF (- 3-15 mM, as highlighted in green). Increasing the bile acid concentration from 0 to 10 mM has no impact on VAN or DAP removal. At a higher concentration of bile acid (> 10 mM), the DAP removal remains unchanged, whereas VAN removal decreases to - 80%. The reduction of VAN removal is possibly a result of the electrostatically driven SEV-bile acid interactions, rendering the amine groups of SEV less accessible for hydrogen bonding with VAN. Although the SOs" : COO" molar ratio of bile acid : DAP is 1 : 1 at - 12 mM of bile acid, the DAP removal decreased by less than 5%. Theoretically, the DAP removal should decrease by - 25%, assuming that the electrostatic interactions in DAP-SEV (1 mol COO" : 1 mol NH3+) are the same as that of bile acid-SEV (1 mol SO3-: 1 mol NH3+).

[0149] FIG. 24 shows the effect of maleic acid concentration on the antibiotic removal. The physiological concentration range of maleic acid in the SIF (- 19 mM in the fasted-state SIF, and up to - 55 mM in the fed-state SIF) is highlighted in blue. By increasing the maleic acid concentration from 0 to - 20 mM, the R for VAN or DAP decreases from - 95% to 75% or 90%, respectively. By further increasing the maleic acid concentration to 50 mM, VAN or DAP removal decreases to 56% or 87%, respectively. Similar to the bile acid, the reduction of VAN removal by increasing maleic acid concentration is possibly a result of SEV-maleic acidelectrostatic interactions, reducing the accessible binding sites (i.e., amine groups) of SEV for interacting with VAN. The reduction of DAP removal may be a result of the competitive binding of maleic acid to SEV. At ~ 10 mM of maleic acid, wherein the molar ratio of carboxyl groups of maleic acid to DAP is 1, the DAP removal of SEV reduces ~ 8%. This implies that DAP binding to SEV involves mechanisms other than electrostatic interactions, such as hydrophobic interactions and hydrogen bonding. The effects of maleic acid and bile acid on the VAN removal are more pronounced compared with DAP. This may be attributed to the higher binding energy (electrostatic interactions) between SEV-maleic acid or SEV-bile acid, dominating the non- electrostatic interactions between SEV and VAN.

[0150] PC, a zwitterionic phospholipid, is one of the main constituents of SIF, which may influence the SEV-VAN and SEV-DAP interactions. FIG. 25 shows the SEV-mediated removal of VAN or DAP at PC concentrations varying from 0.75 to 3.75 mM, i.e., the physiological concentration range from fasted-state SIF to fed-state SIF (highlighted in yellow). As the PC concentration increases to 5 mM, the VAN removal decreases to 70%, while the DAP removal remains ~ 95%. The decreased R for VAN may be a result of the competitive adsorption of PC to SEV. The underlying reason could be the stronger electrostatic interactions between PC and SEV compared with VAN-SEV non-electrostatic interactions. Although the DAP and PC may compete for the cationic SEV, the DAP removal does not change significantly. The unaffected DAP removal may be a result of PC-induced hydrophobic interactions with DAP, leading to PC- DAP aggregation and removal by SEV.

[0151] FIG. 26 shows the VAN or DAP removal (precipitation) without SEV at varying PC concentrations, ranging from 0.5 to 5 mM. The R for VAN is only ~ 5% at 5 mM of PC, whereasDAP R reaches up to ~ 26%, suggesting PC-DAP assembly and precipitation as a result of hydrophobic interactions. The PC-DAP assembly was confirmed via DLS size measurements in our previous study, wherein the PC hydrodynamic size increased from 800 nm to 8000 nm when added to a DAP solution.

[0152] Effect of SIF in the Fasted- or Fed-State on the SEV-Mediated DAP or VAN Removal: To evaluate the antibiotic removal at in vitro conditions that emulate the intestine, combinatorial effects of SIF components on SEV -mediated VAN or DAP removal are investigated. FIGS. 27-30 show SEV (4 mg mL-1) R and qefor VAN or DAP (1 mg mL-1) in the fasted-state SIF or fed-state SIF after 24 h of incubation. FIG. 27 presents the VAN removal at initial VAN concentrations varying from 1 mg mL-1to 20 mg mL-1in the fasted-state SIF or fed- state SIF. At initial VAN concentration of ~ 1 mg mL-1in both body fluids, the R remains ~ 95%; however, at higher initial VAN concentrations ( > 2 mg mL-1), the R decreases as a result of SEV saturation. FIG. 28 shows the SEV qefor VAN versus initial VAN concentrations in the fasted-state SIF or fed-state SIF. The qein fasted-state SIF (1100 ± 36 mg g"1) and fed-state SIF (1200 ± 40 mg g"1) are less than that in ultrapure water (2572 ± 17 mg g"1for VAN, FIG. 14), which is likely due to the competitive adsorption of SIF components, mainly maleic acid.

[0153] FIG. 29 presents the DAP removal at initial DAP concentrations varying from 1 mg mL"1to 20 mg mL-1in the fasted-state SIF or fed-state SIF. The R remains ~ 95% at initial DAP concentrations ranging from 1 mg mL-1to 5 mg mL-1in both body fluids; however, by further increasing the initial DAP concentrations (i.e., > 5 mg mL-1), the R decreases, reaching ~ 60- 70%. FIG. 30 shows the SEV qefor DAP versus initial DAP concentrations in the fasted-state SIF or fed-state SIF. The qein the fasted-state SIF (2300 ± 50 mg g"1) and fed-state SIF (2800 ±18 mg g-1) are less than that in ultrapure water (2857 ± 7 mg g-1for DAP, FIG. 31), likely as a result of SEV saturation with SIF components, such as maleic acid. The non-significant difference in the qein the fasted-state SIF and fed-state SIF implies the feasibility of SEV- mediated VAN or DAP removal, independent of food-induced alterations in the SIF composition.

[0154] In Vitro Assessment of SEV as an Anti-antibiotic: After the SEV-mediated removal of physiologically relevant concentrations of VAN (0.125 to 8 pg mL-1) or DAP (0.125 to 16 pg mL-1), the antimicrobial activities of VAN against VSE / h? or DAP against VRE / m are investigated via the broth microdilution assay. FIGS. 32-34 present the in vitro antibiotic activity of uncaptured VAN or DAP after SEV-mediated adsorption. FIG. 32 shows a stepwise schematic of microdilution assay procedure by which the antibiotic activity of uncaptured VAN or DAP is assessed. Varying doses of SEV are incubated with the antibiotic solutions containing 128 pg mL-1of VAN or 256 pg mL-1of DAP for predetermined periods. After centrifugation, supernatants are diluted using the CAMHB medium to reach the required concentration range and mixed with a known density of bacteria (1.5 x 108CFU mL-1), followed by incubation at 35 °C for 24 h. FIG. 33 presents the bacterial density (ODeoo) following growth in contact with the supernatant collected from SEV-DAP mixtures at varying incubation times. No antibiotic activity is detected against VRE / w after only 5 min of DAP incubation with SEV. This finding agrees with the results of SEV-mediated DAP removal (FIG. 13), implying that 4 mg mL-1of SEV entirely removes DAP from the medium within 5 min, eliminating the antibiotic activity ofDAP in vitro.

[0155] FIG. 34 shows ODeoo following growth in contact with the supernatant collected after incubating VAN with varying doses of SEV, ranging from 20 to 40 mg mL-1, for 4 h. The available antibiotic against VSE / m decreases by increasing SEV dose, giving rise to an increase in bacterial density. No antibiotic activity is detected for VAN after 4 h when SEV dose was 40 mg mL-1. Like DAP, the antibiotic activity of VAN in contact with 4 mg of SEV per mL at varying incubation periods is evaluated (FIG. 35), showing that VAN removal is time-dependent, and the SEV concentration is insufficient for eliminating VAN even after 4 h. The antibiotic activity of VAN after 4 h of incubation with SEV concentrations of 4 - 20 mg mL-1is shown in FIG. 36. Accordingly, the VAN removal is SEV dose dependent, and a higher SEV dose than the experimented range is required to entirely block the VAN activity. FIG. 37 presents ODeoo following growth in contact with the supernatant collected after incubating 40 mg of SEV per mL with VAN for different periods. As incubation time increases, the available antibiotic against VSE / m decreases, increasing the bacterial density. When SEV dose is 40 mg mL-1, no VAN remains in the supernatant after 4 h of incubation, hence no antibiotic activity is observed, resulting in an enhancement in bacterial density. The findings imply that SEV, at an optimal concentration, is able to block the activity of both VAN and DAP at physiologically relevant concentrations in vitro.

[0156] In Vivo Evaluation of SEV Anti-VAN Efficacy: The ability of SEV to block the antimicrobial activity of VAN in vivo is assessed using a mouse model of intestinal E.faecium colonization. Since SEV is administered as sevelamer carbonate (Renvela) in clinical settings, they were used in the in vivo studies. FIG. 38 compares the VAN removal percentage of sevelamer hydrochloride and sevelamer carbonate at varying initial VAN concentrations. At lowinitial VAN concentration (1 mg mL-1), the R ~ 95% for both materials, indicating their similar functionality. FIG. 39 schematically shows the timeline of in vivo assay in which mice were treated for 7 days with ampicillin, followed by inoculation with 108CFU of E.faecium at a 1 : 1000 or 1 : 2.5 ratio of VREfm to VSEfm and the start of twice daily treatment with 30 mg of SEV for 8 days. Beginning one day post inoculation, mice were treated with VAN (60 mg kg'1) for 5 days. At select time points, fecal pellets were collected, and polymerase chain reaction (PCR) was used to quantify the resulting changes in fecal VanA (as a measure of VAN resistance) and E.faecium gene densities in response to VAN treatment with or without SEV. FIG. 40 presents the effects of SEV administration on fecal E. faecium shedding (£. faecium gene copy # per mg of feces) through 14 days post bacterial inoculation. The twice daily SEV treatment has a minimal impact on the density of E. faecium shedding (gene copy # per mg of feces) through 14 days post inoculation relative to untreated controls for either inoculation. FIG. 41 shows cumulative E.faecium shedding, as measured by the area under the fecal density time- series curve (AUC) in FIG. 40. SEV treatment significantly reduced median cumulative E. faecium shedding by ~ 65% (95% confidence interval, CI: 27% - 100%), which did not significantly differ between the two inoculations (p = 0.99). FIG. 42 presents the VanA fecal density (VanA gene copy # mg-1feces) with and without SEV treatment through 14 days post bacterial inoculation. In contrast with E. faecium, VanA fecal density is significantly reduced following VAN treatment in the presence of SEV for both inoculation frequencies, with VanA fecal density being significantly lower in mice inoculated at 1 : 1000 ratio of VREfm to NSEfm relative to those inoculated at a 1 : 2.5 ratio of VRE / h? to VSE / m. The cumulative effects of SEV mediated changes in VanA shedding in response to VAN treatment are shown in FIG. 43,defined as the area under the VanA density time-series curve (AUC). This data demonstrates that the twice daily treatment of mice with 30 mg of SEV is sufficient to reduce median cumulative fecal VanA shedding by 75% (95% CI: 24% - 100%,). The reduction in VanA shedding tends to be greater in mice inoculated at a 1 : 1000 ratio of VRE / m to VSE / m relative to those inoculated at a 1 : 2.5 ratio of VRE / m to VSE / w, though the difference is not statistically significant (p = 0.10).

[0157] As SEV treatment results in a significant reduction in VanA, but not E. faecium fecal shedding, we next ask whether SEV can reduce the frequency of VRE / m following VAN treatment. FIG. 44 shows the effect of SEV treatment on the relative proportion of VanA shed (VanA shed / E. faecium shed) as an indirect measure of changes in VREfm frequency at each time point through 14 days post inoculation. SEV treatment reduces the proportion of VREfm shed at all time points in mice inoculated at a 1 : 1000 ratio of VREfm to VREfm, but not in mice inoculated at a 1 : 2.5 ratio ofVREfm to VSE / bi. FIG. 45 presents the effects of SEV treatment on the VREfm AUC proportion (VanA AUC through 14 days / E. faecium AUC through 14 days) as an indirect measure of cumulative shifts in the frequency of VREfm. Though SEV treatment significantly reduces the cumulative frequency ofVREfm shedding in mice inoculated at a 1 : 1000 ratio ofVREfm to VSE fm (p = 0.03), no reduction in the cumulative frequency of VREfm is observed in mice inoculated at a ratio of 1 : 2.5 (p = 0.69) or in aggregate (p = 0.35). Taken together, these findings show that SEV inhibits the antibiotic activity of VAN in vivo, with SEV treatment significantly reducingVREfm shedding following VAN exposure.

[0158] Based on our conducted evaluation work discussed herein, it has been shown that embodiments of our process can account for use of last-line antibiotics such as VAN and DAPthat may enter the GI tract of a patient via biliary excretion wherein they lack therapeutic value so that such components can be effectively removed to avoid the presence of these compounds driving development of bacterial resistance to them in Gl-dwelling opportunistic bacteria. For example, our experimentation showed that use of SEV as an antibacterial removal agent can be used as an adjuvant therapy to eliminate off-target antibiotics from the GI tract and prevent AMR.

[0159] Our experimental results showed that SEV adsorbs low concentrations (i.e. , 1 mg mL-1) of negatively charged lipopeptide DAP within seconds and positively charged glycopeptide VAN within 2 h with a capacity above 2500 mg g-1. DAP adsorption by SEV is mainly controlled by electrostatic interactions, whereas VAN adsorption may be governed by non- covalent interactions, such as hydrogen bonding. The time-dependent adsorption was established for both VAN and DAP, corresponding to the molecular diffusion of antibiotics inside porous SEV. In the FaSSIF or FeSSIF, the removal of 1 mg mL-1antibiotics by SEV (4 mg mL-1) remains unchanged (~ 95%). The antibiotic activity of VAN or DAP was blocked by 40 mg mL-1SEV after 4 h or 4 mg mL-1SEV after 5 min, respectively. In mouse experiments, SEV effectively reduced the enrichment of VAN resistance following systemic VAN treatment, demonstrating the potential of this approach in vivo. We envision that the adjuvant therapy with dual-functional SEV may serve as a ground-breaking treatment strategy to prevent VAN- and DAP-induced resistance evolutions.

[0160] Our experimental work shows that the use of an antibiotic removal agent or antibiotic deactivation agent (e.g. SEV) may be a suitable treatment for providing to a patient to effectively remove VAN from a patient’s GI tract, but that it can remove both VAN and DAP from thepatient GI tract. Our experimental work supports our belief that embodiments can be employed in conjunction with the administration of IV antibacterial injections to help eliminate excess antibacterial compounds that may be present in the patient’s GI tract as a consequence of the treatment without hindering the treatment’s ability to treat the bacterial infection in a target location of the patient. Embodiments of the process can facilitate improved health outcomes by avoiding the patient undergoing negative consequence of antibacterial compounds interfering with the patient’s gut bacteria while also avoiding the evolution of antibacterial resistant bacteria within the patients 1G tract.

[0161] It should be understood that the disclosure of a range of values is a disclosure of every numerical value within that range, including the end points. It should also be appreciated that some components, features, and / or configurations may be described in connection with only one particular embodiment, but these same components, features, and / or configurations can be applied or used with many other embodiments and should be considered applicable to the other embodiments, unless stated otherwise or unless such a component, feature, and / or configuration is technically impossible to use with the other embodiment. Thus, the components, features, and / or configurations of the various embodiments can be combined together in any manner and such combinations are expressly contemplated and disclosed by this statement.

[0162] It will be apparent to those skilled in the art that numerous modifications and variations of the described examples and embodiments are possible considering the above teachings of the disclosure. The disclosed examples and embodiments are presented for purposes of illustration only. Other alternate embodiments may include some or all of the features disclosed herein.Therefore, it is the intent to cover all such modifications and alternate embodiments as may come within the true scope of this invention, which is to be given the full breadth thereof.

[0163] It should be understood that modifications to the embodiments disclosed herein can be made to meet a particular set of design criteria. Therefore, while certain exemplary embodiments of the apparatus and methods of using and making the same disclosed herein have been discussed and illustrated, it is to be distinctly understood that the invention is not limited thereto but may be otherwise variously embodied and practiced within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A process for removing one or more antibacterial compounds from a gastrointestinal (GI) tract of a patient, comprising: administering at least one antibiotic removal agent or at least one antibiotic deactivation agent to the patient for digestion so that the at least one antibiotic removal agent or the at least one antibiotic deactivation agent is present in the GI tract of the patient; the at least one antibiotic removal agent or the or at least one antibiotic deactivation agent effectively removing the one or more antibacterial compounds present in the GI tract.

2. The process of claim 1, wherein the one or more antibacterial compounds include Vancomycin (VAN).

3. The process of claim 2, wherein the one or more antibacterial compound also includes daptomycin (DAP) and / or linezolid.

4. The process of claim 1, wherein the at least one antibiotic removal agent or the or at least one antibiotic deactivation agent effectively removing the one or more antibacterial compounds present in the GI tract includes the at least one antibiotic removal agent interacting with the one or more antibacterial compounds present in the GI tract such that one or more antibacterial compounds present in the GI tract are non-bioreactive with bacteria within the GI tract.

5. The process of claim 1, wherein the at least one antibiotic removal agent or the at least one antibiotic deactivation agent is comprised of sevelamer (SEV).

6. The process of claim 1, wherein the at least one antibiotic removal agent or the at least one antibiotic deactivation agent is comprised of patiromer, sodium polystyrene sulfonate, sodium zirconium cyclosilicate, sodium polystyrene sulfonate, sodium polystyrene sulfonate, colestipol, tolevamer, polacrilex resin (Amberlite IRP64), polacrilin potassium (Amberlite IRP88), colesevelam, sevelamer (SEV), magnesium-containing compounds, calcium-containing compounds, iron-containing compounds, zinc-containing compounds, antacids containing aluminum or magnesium, proton pump inhibitors, or combinations thereof.

7. The process of claim 1, comprising: treating the patient with the one or more antibacterial compounds to treat an infectious disease in the patient.

8. The process of claim 7, wherein the infectious disease is a bacterial infection that is not located in the GI tract.

9. The process of claim 1, wherein the administering of the at least one antibiotic removal agent or the at least one antibiotic deactivation agent to the patient for the patient to digest so that the at least one antibiotic removal agent or the at least one antibiotic deactivation agent is present in the GI tract of the patient comprises:feeding the patient the at least one antibiotic removal agent or the at least one antibiotic deactivation agent such that the at least one antibiotic removal agent or the at least one antibiotic deactivation agent is orally consumed by the patient.

10. The process of claim 1, wherein the administering of the at least one antibiotic removal agent or the at least one antibiotic deactivation agent to the patient for the patient to digest so that the at least one antibiotic removal agent or the at least one antibiotic deactivation agent is present in the G1 tract of the patient comprises: feeding the patient the at least one antibiotic removal agent or the at least one antibiotic deactivation agent periodically while the patient undergoes a treatment for an infection that includes multiple injections of the one or more antibacterial compounds into the patient at different spaced apart intervals.

11. The process of claim 1 , wherein the administering is performed periodically different times a day for a number of days.

12. The process of claim 1, wherein the at least one antibiotic removal agent or the at least one antibiotic deactivation agent effectively removes the one or more antibacterial compounds present in the GI tract such that bacteria within the GI tract is unaffected by the antibacterial compounds.

13. The process of claim 1, wherein the process is performed in a hospital, a care facility, or a farm.

14. The process of claim 1, comprising: injecting the patient with and / or orally administering to the patient the one or more antibacterial compounds to treat an infectious disease in the patient.

15. The process of claim 1, comprising: injecting the patient with the one or more antibacterial compounds to treat an infectious disease in the patient; wherein the infectious disease is a bacterial infection that is not located in the GI tract; and wherein the administering of the at least one antibiotic removal agent or the at least one antibiotic deactivation agent to the patient for the patient to digest so that the at least one antibiotic removal agent or the at least one antibiotic deactivation agent is present in the GI tract of the patient comprises: feeding the patient the at least one antibiotic removal agent or the at least one antibiotic deactivation agent such that the at least one antibiotic removal agent is orally consumed by the patient.

16. The process of claim 1, wherein the at least one antibiotic removal agent or the at least one antibiotic deactivation agent is comprised of a polymeric material.

17. The process of claim 1, wherein the at least one antibiotic removal agent or the at least one antibiotic deactivation agent is comprised of at least one anion exchange polymeric material.

18. The process of claim 1, wherein the at least one antibiotic removal agent or the at least one antibiotic deactivation agent is comprised of at least one cation exchange polymeric material.

19. The process of claim 1, wherein the at least one antibiotic removal agent or the at least one antibiotic deactivation agent is comprised of a nanoparticle and / or microparticle.

20. The process of claim 1, wherein the at least one antibiotic removal agent or the at least one antibiotic deactivation agent is comprised of a small molecule.

21. The process of claim 1, wherein the at least one antibiotic removal agent or the at least one antibiotic deactivation agent is comprised of an organic and / or inorganic material.

22. The process of claim 1, wherein the at least one antibiotic removal agent or the or at least one antibiotic deactivation agent is configured protect microbiome present in the GI tract of the patient.

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