Biopolymeric Anti-antibiotics and methods for making and using the same

Abstract

A process and system for removal of antibiotic compounds from a patient's body can include having a patient digest an antibiotic removal agent or an antibiotic deactivation agent. The agent can include anionic hairy cellulose nanocrystals (AHCNC) hybridized with a resin, such as cholestyramine (CHA). The agent can pass through the patient's digestive system to remove antibiotic compounds that may reside therein as a consequence of an antibacterial treatment provided to the patient. Removal of the antibiotic compounds from the patient's digestive system can avoid evolution of antimicrobial resistance.

Description

Atty. Ref. No. 0073605-000956BIOPOLYMERIC ANTI-ANTIBIOTICS AND METHODS FOR MAKING AND USING THE SAMECROSS-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 Hatch Act Project No. PEN04769 awarded by the United States Department of Agriculture. The Government has certain rights in the invention.FIELD

[0003] Embodiments relate to processes and apparatus configured to reduce, hinder, or avoid the growth or development of drug resistant bacteria and / or the protection of drug sensitive bacteria. Embodiments can be adapted to help block or avoid antibioticresistant bacterial growth within the body of a patient, for example. Embodiments can be adapted to help protect or promote antibiotic-sensitive bacterial growth within the body of a patient, for example. Embodiments can be adapted to help protect the microbiome within the body of a patient, for example.BACKGROUND

[0004] To combat bacterial infections in humans, antibiotics have extensively been used to save millions of lives since the 20thcentury. Vancomycin (VAN), a glycopeptide antibiotic, isolated from the fermentation of a soil actinomycete, has been a key drug in treating Gram-positive pathogen infections, such as methicillin-resistant Staphylococcus aureus (MRSA). VAN inhibits bacterial cell wall biosynthesis through binding to the D-alanyl-D-alanine moieties of peptidoglycan layer in the bacteria cell wall. This bindingAtty. Ref. No. 0073605-000956occurs via five hydrogen bonds between VAN heptapeptide and the D-alanyl-D-alanine, as confirmed by the NMR spectroscopy. Accordingly, VAN has been one of the most effective regimens for Gram-positive pathogen infections.

[0005] In clinical treatments, VAN is usually administered intravenously to patients infected by Gram-positive pathogens. Excess VAN in the bloodstream is mainly eliminated by the kidneys; however, biliary excretion leads to the bioavailability of ~ 5 -10% of intravenous VAN in the intestines, which has been confirmed by measuring up to 100 pg of VAN per mL in human stools. The intestine-exposed VAN may drive the VAN-susceptible Enterococcus faecium (VSEfrnj, a common colonizer, to gain resistance. VAN-resistant E. faecium (\ / REfm) may enter the bloodstream via moving across the intestinal lining and passing through the liver, which potentially causes infective endocarditis when contacting the heart. Additionally, environmental fecal exposure may lead to the VRE transmission to other patients via contaminating the medical equipment or communal surfaces in hospitals.SUMMARY

[0006] We hypothesize that capturing off-target VAN in the Gl tract may prevent the evolution and transmission of VREs. So far, a few approaches have been investigated to protect the gut microbiome via capturing or deactivating antibiotics, including the oral administration of D-alanyl-D-alanine-functionalized polyethylene glycol microparticles and D-alanyl-D-alanine-based antagonists. However, the building blocks of D-alanyl-D-alanine-functionalized microparticles comprise L-amino acids, in addition to D-alanyl-D-alanine, which are susceptible to enzymatic degradation in the Gl tract. Additionally, in vivo studies were conducted on mice orally treated with high concentrations of VAN, conditions that do not reflect clinical scenarios wherein VAN is administered intravenously. Among the peptide libraries mimicking the bacterial VAN binding site, i.e., D-alanyl-D-alanine, a few analogs could neutralize VAN activity against Enterococci, but only at high peptide:VAN molar ratios. For example, acetyl-lysine(acetyl)- D-alanyl-D-alanine was effective at a peptide:VAN ratio of 28:1. In addition, the analogs were moreAtty. Ref. No. 0073605-000956effective at high VAN concentrations (e.g., 32 pg mL-1), compared with low VAN concentrations (e.g., 8 pg mb1), which is closer to the physiological Gl-exposed VAN concentration. Accordingly, translating the current peptide-enabled analogs to in vivo applications is challenging.

[0007] We have developed a novel anti-VAN biomaterial via hybridizing hairy cellulose nanocrystals with a Food and Drug Administration (FDA)-approved resin to remove the excess antibiotic from the Gl tract before it impacts bacteria and selects for resistance. In vitro studies show that VAN removal is regulated by electrostatic interactions via a time-dependent diffusion-controlled process that is not significantly influenced by the physiological pH, ionic strength, or the components of simulated intestinal fluid. Aligned with the in vitro findings, the oral administration of anti-VAN adjuvant may effectively sequester VAN in the murine Gl tract and prevented the VAN resistance enrichment following the VAN treatment of E. faecium colonized mice. The anti-VAN biomaterial may protect intravenous VAN, which can be a step forward in addressing the global threat of antimicrobial resistance.

[0008] In an exemplary embodiment, an antibiotic removal or deactivation agent comprises hairy cellulose nanocrystals; a particulate resin; and optionally, a functionalized biopolymer, wherein the hairy cellulose nanocrystals are immobilized on the particulate resin.

[0009] In some embodiments, the particulate resin is comprised of cholestyramine, 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.

[0010] In some embodiments, the particulate resin is cholestyramine.Atty. Ref. No. 0073605-000956

[0011] In some embodiments, the hairy cellulose nanocrystals comprise cellulose bodies bearing cellulose chains protruding therefrom, wherein the cellulose chains are functionalized with anionic groups.

[0012] In some embodiments, the anionic groups comprise carboxylate groups.

[0013] In some embodiments, the hairy cellulose nanocrystals comprise cellulose bodies bearing cellulose chains protruding therefrom, wherein the cellulose chains are functionalized with cationic groups.

[0014] In some embodiments, the hairy cellulose nanocrystals comprise cellulose bodies bearing cellulose chains protruding therefrom, wherein the cellulose chains are functionalized with amphiphilic groups.

[0015] In some embodiments, the hairy cellulose nanocrystals comprise cellulose bodies bearing cellulose chains protruding therefrom, wherein the cellulose chains are functionalized with zwitterionic groups.

[0016] In some embodiments, the hairy cellulose nanocrystals comprise cellulose bodies bearing cellulose chains protruding therefrom, wherein the cellulose chains are functionalized with a combination of electrically neutral anionic, cationic, zwitterionic, and / or amphiphilic groups.

[0017] In some embodiments, the functionalized biopolymer comprises polysaccharide, protein, polynucleotides, and / or polypeptides, wherein the biopolymer is functionalized with a combination of electrically neutral anionic, cationic, zwitterionic, and / or amphiphilic groups.

[0018] In some embodiments, the agent is configured to be administered to a gastrointestinal (Gl) tract of a patient, such that when the Gl tract includes one or more antibiotic compounds, the agent is configured to effectively remove the one or more antibiotic compounds.

[0019] In some embodiments, the one or more antibiotic compounds include B-lactams, aminoglycosides, chloramphenicol, glycopeptides, quinolones, oxazolidinones, sulfonamides, tetracyclines, macrolides, ansamycins, streptogramins, lipopeptides, or combinations thereof.Atty. Ref. No. 0073605-000956

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

[0021] In an exemplary embodiment, an antibiotic removal or deactivation agent comprises a functionalized biopolymer, wherein the functionalized biopolymer comprises polysaccharide, protein, polynucleotides, and / or polypeptides, wherein the polymer is functionalized with a combination of electrically neutral anionic, cationic, zwitterionic, and / or amphiphilic groups.

[0022] In some embodiments, the agent is configured to be administered to a gastrointestinal (Gl) tract of a patient, such that when the Gl tract includes one or more antibiotic compounds, the agent is configured to effectively remove the one or more antibiotic compounds.

[0023] In some embodiments, the one or more antibiotic compounds include B-lactams, aminoglycosides, chloramphenicol, glycopeptides, quinolones, oxazolidinones, sulfonamides, tetracyclines, macrolides, ansamycins, streptogramins, lipopeptides, or combinations thereof.

[0024] In some embodiments, the one or more antibiotic compounds include Vancomycin (VAN).

[0025] In an exemplary embodiment, a process for removing one or more antibiotic compounds from a gastrointestinal (Gl) tract of a patient comprises administering an antibiotic removal or deactivation agent to the patient for digestion so that the antibiotic removal agent or the antibiotic deactivation agent is present in the Gl tract of the patient, wherein the antibiotic removal or deactivation agent effectively removes the one or more antibiotic compounds present in the Gl tract, and wherein the antibiotic removal or deactivation agent comprises hairy cellulose nanocrystals immobilized on a particulate resin.

[0026] In some embodiments, the particulate resin is comprised of cholestyramine, patiromer, sodium polystyrene sulfonate, sodium zirconium cyclosilicate, sodium polystyrene sulfonate, sodium polystyrene sulfonate, colestipol, tolevamer, polacrilex resin (Amberlite IRP64), polacrilin potassium (Amberlite IRP88), colesevelam,Atty. Ref. No. 0073605-000956sevelamer (SEV), magnesium-containing compounds, calcium-containing compounds, iron-containing compounds, zinc-containing compounds, antacids containing aluminum or magnesium, proton pump inhibitors, or combinations thereof.

[0027] In some embodiments, the particulate resin comprises cholestyramine.

[0028] In some embodiments, the one or more antibiotic compounds include B-lactams, aminoglycosides, chloramphenicol, glycopeptides, quinolones, oxazolidinones, sulfonamides, tetracyclines, macrolides, ansamycins, streptogramins, lipopeptides, or combinations thereof.

[0029] In some embodiments, the one or more antibiotic compounds include Vancomycin (VAN).

[0030] In some embodiments, the antibiotic removal or deactivation agent effectively removing the one or more antibiotic compounds present in the Gl tract includes the antibiotic removal or deactivation agent interacting with the one or more antibiotic compounds present in the Gl tract such that one or more antibacterial compounds present in the Gl tract are non-bioreactive with bacteria within the Gl tract.

[0031] In some embodiments, the process further comprises treating the patient with the one or more antibiotic compounds to treat an infectious disease in the patient.

[0032] In some embodiments, the infectious disease is a bacterial infection that is not located in the Gl tract.

[0033] In some embodiments, the infectious disease is a bacterial infection that is located in the Gl tract.

[0034] In some embodiments, the process further comprises treating the patient with the one or more antibiotic compounds to treat a non-infectious disease in the patient.

[0035] In some embodiments, the non-infectious disease is a bacterial infection that is not located in the Gl tract.

[0036] In some embodiments, the non-infectious disease is a bacterial infection that is located in the Gl tract.

[0037] In some embodiments, the administering of the antibiotic removal or deactivation agent to the patient for the patient to digest so that the antibiotic removal or deactivationAtty. Ref. No. 0073605-000956agent is present in the Gl tract of the patient comprises feeding the patient the antibiotic removal or deactivation agent such that the antibiotic removal or deactivation agent is orally consumed by the patient.

[0038] In some embodiments, the administering of the antibiotic removal or deactivation agent to the patient for the patient to digest so that the antibiotic removal or deactivation agent is present in the Gl tract of the patient comprises feeding the patient the antibiotic removal or deactivation agent periodically while the patient undergoes a treatment for an infection that includes multiple injections of the one or more antibiotic compounds into the patient at different spaced apart intervals.

[0039] In some embodiments, the administering is performed periodically different times a day for a number of days.

[0040] In some embodiments, the antibiotic removal or deactivation agent effectively removes the one or more antibiotic compounds present in the Gl tract such that bacteria within the Gl tract is unaffected by the antibiotic compounds.

[0041] In some embodiments, the antibiotic removal or deactivation agent effectively removes the one or more antibiotic compounds present in the Gl tract to reduce, hinder, or avoid the growth or development of drug resistant bacteria and / or the protection of drug sensitive bacteria.

[0042] In some embodiments, the antibiotic removal or deactivation agent effectively removes the one or more antibiotic compounds present in the Gl tract to block or avoid antibiotic-resistant bacterial growth within the body.

[0043] In some embodiments, the antibiotic removal or deactivation agent effectively removes the one or more antibiotic compounds present in the Gl tract to help protect or promote antibiotic-sensitive bacterial growth within the body.

[0044] In some embodiments, the antibiotic removal or deactivation agent effectively removes the one or more antibiotic compounds present in the Gl tract to help protect the microbiome within the body.

[0045] In an exemplary embodiment, a process for removing one or more antibiotic compounds from a gastrointestinal (Gl) tract of a patient comprises administering anAtty. Ref. No. 0073605-000956antibiotic removal or deactivation agent to the patient for digestion so that the antibiotic removal agent or the antibiotic deactivation agent is present in the Gl tract of the patient, wherein the antibiotic removal or deactivation agent effectively removes the one or more antibiotic compounds present in the Gl tract, and wherein the antibiotic removal or deactivation agent comprises a functionalized biopolymer, wherein the functionalized biopolymer comprises polysaccharide, protein, polynucleotides, and / or polypeptides, wherein the polymer is functionalized with a combination of electrically neutral anionic, cationic, zwitterionic, and / or amphiphilic groups.

[0046] In some embodiments, the particulate resin is comprised of cholestyramine, 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.

[0047] In some embodiments, the particulate resin comprises cholestyramine.

[0048] In some embodiments, the one or more antibiotic compounds include B-lactams, aminoglycosides, chloramphenicol, glycopeptides, quinolones, oxazolidinones, sulfonamides, tetracyclines, macrolides, ansamycins, streptogramins, lipopeptides, or combinations thereof.

[0049] In some embodiments, the one or more antibiotic compounds include Vancomycin (VAN).

[0050] In some embodiments, the antibiotic removal or deactivation agent effectively removing the one or more antibiotic compounds present in the Gl tract includes the antibiotic removal or deactivation agent interacting with the one or more antibiotic compounds present in the Gl tract such that one or more antibacterial compounds present in the Gl tract are non-bioreactive with bacteria within the Gl tract.

[0051] In some embodiments, the process further comprises treating the patient with the one or more antibiotic compounds to treat an infectious disease in the patient.Atty. Ref. No. 0073605-000956

[0052] In some embodiments, the infectious disease is a bacterial infection that is not located in the Gl tract.

[0053] In some embodiments, the infectious disease is a bacterial infection that is located in the Gl tract.

[0054] In some embodiments, the process further comprises treating the patient with the one or more antibiotic compounds to treat a non-infectious disease in the patient.

[0055] In some embodiments, the non-infectious disease is a bacterial infection that is not located in the Gl tract.

[0056] In some embodiments, the non-infectious disease is a bacterial infection that is located in the Gl tract.

[0057] In some embodiments, the administering of the antibiotic removal or deactivation agent to the patient for the patient to digest so that the antibiotic removal or deactivation agent is present in the Gl tract of the patient comprises feeding the patient the antibiotic removal or deactivation agent such that the antibiotic removal or deactivation agent is orally consumed by the patient.

[0058] In some embodiments, the administering of the antibiotic removal or deactivation agent to the patient for the patient to digest so that the antibiotic removal or deactivation agent is present in the Gl tract of the patient comprises feeding the patient the antibiotic removal or deactivation agent periodically while the patient undergoes a treatment for an infection that includes multiple injections of the one or more antibiotic compounds into the patient at different spaced apart intervals.

[0059] In some embodiments, the administering is performed periodically different times a day for a number of days.

[0060] In some embodiments, the antibiotic removal or deactivation agent effectively removes the one or more antibiotic compounds present in the Gl tract such that bacteria within the Gl tract is unaffected by the antibiotic compounds.

[0061] In some embodiments, the antibiotic removal or deactivation agent effectively removes the one or more antibiotic compounds present in the Gl tract to reduce, hinder,Atty. Ref. No. 0073605-000956or avoid the growth or development of drug resistant bacteria and / or the protection of drug sensitive bacteria.

[0062] In some embodiments, the antibiotic removal or deactivation agent effectively removes the one or more antibiotic compounds present in the Gl tract to block or avoid antibiotic-resistant bacterial growth within the body.

[0063] In some embodiments, the antibiotic removal or deactivation agent effectively removes the one or more antibiotic compounds present in the Gl tract to help protect or promote antibiotic-sensitive bacterial growth within the body.

[0064] In some embodiments, the antibiotic removal or deactivation agent effectively removes the one or more antibiotic compounds present in the Gl tract to help protect the microbiome within the body.

[0065] 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

[0066] 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.

[0067] FIG. 1 is a schematic illustration of an exemplary embodiment of out antiantibiotic process. The removal of intestine-exposed antibiotics using anti-antibiotics may prevent the emergence of resistant bacteria; however, in the absence of any antiantibiotics, antibiotic resistance evolution following intravenous VAN injection and excretion into the intestines may lead to the emergence of resistant bacteria that may enter the bloodstream and cause infections.Atty. Ref. No. 0073605-000956

[0068] FIG. 2 is a schematic illustration of an exemplary process for nanoengineering of cellulose fibrils via successive oxidation reactions, yielding anionic hairy cellulose nanocrystals (AHCNC).

[0069] FIG. 3 is a representative atomic force microscopy (AFM) image of AHCNC, showing their needle-like crystalline body.

[0070] FIG. 4 is a graph illustrating an aldehyde content of dialdehyde-modified cellulose (DAMC). Titrations were performed using a 10 mM NaOH solution.

[0071] FIG. 5 is a graph illustrating a carboxylate content of AHCNC. Titrations were performed using a 10 mM NaOH solution.

[0072] FIG. 6 is a graph illustrating an AHCNC-mediated vancomycin (VAN) (Co, VAN — 6 mg mL-1) removal percentage (F?) and capacity (ge) versus incubation time. AHCNC dispersion concentration and total volume were 0.25 mg mL-1and 1 mL, respectively. pH was adjusted to 6.5 using a NaOH solution (0.5 M).

[0073] FIG. 7 is a graph illustrating R at varying Co, VAN. AHCNC dispersion concentration and total volume were 0.25 mg mL-1and 1 mL, respectively. pH was adjusted to 6.5 using a NaOH solution (0.5 M).

[0074] FIG. 8 is a graph illustrating qeat varying equilibrium VAN concentrations (Ce.vAN). AHCNC dispersion concentration and total volume were 0.25 mg mL-1and 1 mL, respectively. pH was adjusted to 6.5 using a NaOH solution (0.5 M).

[0075] FIG. 9 is a graph illustrating ^-potential of AHCNC (0.1% w / v) at varying Co, AN, showing a decrease by increasing Co, VAN as a result of electrostatic interactions between AHCNC and VAN, causing charge neutralization. The schematics of nanorods represent AHCNC, and the clusters are VAN molecules, showing the saturation of AHCNC carboxylate groups on the protruding hairs by increasing Co, VAN. pH was adjusted to 6.5 using a NaOH solution (0.5 M).

[0076] FIG. 10 is a graph illustrating hydrodynamic equivalent size of AHCNC (0.1% w / v) at varying Co, VAN. The schematics of nanorods represent AHCNC, and the clusters are VAN molecules, showing the saturation of AHCNC carboxylate groups on theAtty. Ref. No. 0073605-000956protruding hairs by increasing Co, VAN. pH was adjusted to 6.5 using a NaOH solution (0.5 M).

[0077] FIG. 11 is a schematic illustration of the effect of VAN on AHCNC colloidal behavior at an initial VAN concentration (Co, N) below or above the charge stoichiometry ratio.

[0078] FIG. 12 is a schematic illustration of the formation of AHCHC-CHA antiantibiotics via electrostatic interactions.

[0079] FIG. 13 shows optical microscopy images of cholestyramine (CHA) and AHCNC-CHA particles sedimented in ultrapure water.

[0080] FIG. 14 is a graph illustrating the effect of AHCNC-to-CHA mass ratio, shown based on varying molar ratios (MR) of AHCNC COO-: CHA N+(CH3)3, on the AHCNC-CHA carboxylate group content and the mass ratio of adsorbed AHCNC (WA, AHCNC) with respect to CHA mass (mcHA).

[0081] FIG. 15 is a schematic illustration of VAN chemical structure and its removal process by AHCNC-CHA anti-antibiotics.

[0082] FIG. 16 is a graph illustrating VAN removal percentage (R) of AHCNC-CHA with varying formulations, indicating R ~ 95% at the AHCNC-CHA MR above 6, bearing the highest carboxylate group content to interact with VAN amine groups.

[0083] FIG. 17 is a graph illustrating R for varying AHCNC-CHA formulations after 4 h of VAN incubation with AHCNC-CHA in aqueous solutions with varying pH, showing a decrease in R by decreasing the pH.

[0084] FIG. 18 is a graph illustrating mass ratio of desorbed AHCNC mo, AHCNC) from AHCNC-CHA with respect to AHCNC-CHA mass ( / T?AHCNC-CHA) after 6 h of incubation in aqueous solutions with varying pH. The AHCNC-CHA formulated with varying COO' : (N+(CH3)3) MR are shown as AHCNC-CHA-MRn (n = 2, 4, 6, 8, 12).

[0085] FIG. 19 is a graph illustrating VAN (1 mg mL'1) removal percentage (R) of AHCNC-CHA anti-antibiotic at varying doses after 6 h of AHCNC-CHA pre-removal incubation at pH - 1.5. The data within the black dashed bracket shows that theAtty. Ref. No. 0073605-000956equilibrium VAN concentration was lower than the detection limit of UV-vis spectrophotometer, thus R ~ 95% is likely underestimated.

[0086] FIG. 20 shows scanning electron microscope (SEM) images of CHA and AHCNC-CHA.

[0087] FIG. 21 is a table including surface area, pore volume, and average pore diameter of CHA and AHCNC-CHA.

[0088] FIG. 22 is a ATR-FTIR spectra of AHCNC, CHA, and AHCNC-CHA.

[0089] FIG. 23 is a graph illustrating R versus incubation time, showing a timedependent VAN adsorption process as a result of AHCNC-CHA porous structure.AHCNC-CHA represents AHCNC-CHA-MR6. The data within the dashed brackets show that the equilibrium VAN concentration was lower than the detection limit of UV-vis spectrophotometer, thus R - 95% is likely underestimated.

[0090] FIG. 24 is a graph illustrating R versus AHCNC-CHA dose, showing that VAN removal is dose-dependent. AHCNC-CHA represents AHCNC-CHA-MR6. The data within the dashed brackets show that the equilibrium VAN concentration was lower than the detection limit of UV-vis spectrophotometer, thus R ~ 95% is likely underestimated.

[0091] FIG. 25 is a graph illustrating the ratio of mo, AHCNC to ATJAHCNC-CHA after incubating AHCNC-CHA in the SGF for varying periods. AHCNC-CHA represents AHCNC-CHA-MR6.

[0092] FIG. 26 is a graph illustrating R of AHCNC-CHA after 4 h of incubation in SGF versus varying AHCNC-CHA doses, indicating a decrease in R at 20 mg mb1of AHCNC-CHA, which is compensated by increasing AHCNC-CHA dose. In all experiments, Co, VAN = 1 mg mL-1. AHCNC-CHA represents AHCNC-CHA-MR6. The data within the dashed brackets show that the equilibrium VAN concentration was lower than the detection limit of UV-vis spectrophotometer, thus R ~ 95% is likely underestimated.

[0093] FIG. 27 is a graph illustrating the effect of initial VAN concentration (Co, VAN) on VAN removal percentage (R) and capacity (qe) of AHCNC-CHA after 4 h of incubation. AHCNC-CHA dose was 20 mg ml_‘1, and the total sample volume was 1 mL.Atty. Ref. No. 0073605-000956

[0094] FIG. 28 is a graph illustrating R at varying pH (Co, VAN—1 mg mL1). AHCNC-CHA dose was 20 mg mb1, and the total sample volume was 1 mL.

[0095] FIG. 29 is a schematic illustration and table showing the chemical structure of VAN, the pKa values of its major functional groups, and the net charge of VAN and AHCNC at varying pH.

[0096] FIG. 30 is a graph illustrating ^-potentials of VAN and AHCNC at varying pH. Co, VAN = 1 mg mL'1and AHCNC dose = mg mL-1.

[0097] FIG. 31 is a graph illustrating hydrodynamic equivalent size of VAN and AHCNC at varying pH. Co, VAN = 1 mg mL'1and AHCNC dose = mg mL-1.

[0098] FIG. 32 is a graph illustrating Rat varying Na+concentrations (Co, VAN = 1 mg mL'1). AHCNC-CHA dose was 20 mg mL'1, and the total sample volume was 1 mL. The data within the dashed bracket show that the equilibrium VAN concentration was lower than the detection limit of UV-vis spectrophotometer, thus R ~ 95% is likely underestimated.

[0099] FIG. 33 is a graph illustrating) ^-potentials of VAN and AHCNC at varying Na+concentrations. Co, VAN = 1 mg mL'1and AHCNC dose = mg mL'1.

[0100] Fl G. 34 is a graph illustrating hydrodynamic equivalent size of VAN and AHCNC at varying Na+concentrations. Co, VAN = 1 mg mL'1and AHCNC dose = mg mL'1.

[0101] Fl G. 35 is a graph illustrating R at varying Ca2+concentrations (Co, VAN — 1 mg mL-1). AHCNC-CHA dose was 20 mg mL-1, and the total sample volume was 1 mL. The data within the dashed bracket show that the equilibrium VAN concentration was lower than the detection limit of UV-vis spectrophotometer, thus R ~ 95% is likely underestimated.

[0102] Fl G. 36 is a graph illustrating ^-potentials of VAN and AHCNC at varying Ca2+concentrations. Co, VAN = 1 mg mL'1and AHCNC dose = mg mL'1.

[0103] FIG. 37 is a graph illustrating hydrodynamic equivalent size of VAN and AHCNC at varying Ca2+concentrations. Co, VAN = 1 mg mL'1and AHCNC dose = mg mL'Atty. Ref. No. 0073605-000956

[0104] FIG. 38 is a graph illustrating R at 5 mM of Ca2+and varying AHCNC-CHA doses. Co, VAN = 1 mg mL’1. The data within the dashed bracket show that the equilibrium VAN concentration was lower than the detection limit of UV-vis spectrophotometer, thus R ~ 95% is likely underestimated.

[0105] FIG. 39 is a graph illustrating AHCNC-CHA-mediated VAN (Co, VAN = 1 mg mb1) removal percentage (F?) at varying concentrations of bile acid. AHCNC-CHA dose was 20 mg mL’1, and total sample volume = 1 mL. In the graph, BA stands for bile acid. The data within the dashed bracket show that the equilibrium VAN concentration was lower than the detection limit of the UV-vis spectrophotometer, thus R ~ 95% is likely underestimated.

[0106] Fl G. 40 is a graph illustrating AHCNC-CHA-mediated VAN (Co, VAN — 1 mg mL-1) removal percentage (F?) at varying concentrations of maleic acid. AHCNC-CHA dose was 20 mg mL’1, and total sample volume = 1 mL. In the graph, MA stands for maleic acid. The data within the dashed bracket show that the equilibrium VAN concentration was lower than the detection limit of the UV-vis spectrophotometer, thus R ~ 95% is likely underestimated.

[0107] Fl G. 41 is a graph illustrating R at 20 mM of maleic acid (MA) and varying AHCNC-CHA doses. Co, VAN = 1 mg mL’1. The data within the dashed bracket show that the equilibrium VAN concentration was lower than the detection limit of UV-vis spectrophotometer, thus R ~- 95% is likely underestimated.

[0108] Fl G. 42 is a graph illustrating AHCNC-CHA-mediated VAN (Co, AN — 1 mg mL’1) removal percentage (F?) at varying concentrations of phosphatidylcholine (PC). AHCNC-CHA dose was 20 mg mL’1, and total sample volume = 1 mL. The data within the dashed bracket show that the equilibrium VAN concentration was lower than the detection limit of the UV-vis spectrophotometer, thus R ~ 95% is likely underestimated.

[0109] FIG. 43 is a graph illustrating R at varying Co, AN in the FaSSIF or FeSSIF. AHCNC-CHA dose was 20 mg mL’1, and total sample volume = 1 mL.Atty. Ref. No. 0073605-000956

[0110] Fl G. 44 is a graph illustrating removal capacity (ge) at varying Co, AN in the FaSSIF or FeSSIF. AHCNC-CHA dose was 20 mg mb1, and total sample volume = 1 mL.

[0111] FIG. 45 is a graph illustrating the optical density (ODeoo) of bacteria (VSEfrn), cultured in the supernatant of VAN after incubation with AHCNC, CHA, or AHCNC-CHA for 4 h. The concentration of all sorbents (AHCNC, CHA, or AHCNC-CHA) was fixed at 20 mg mL-1. CHA, AHCNC, and AHCNC-CHA control groups are VAN-free.

[0112] FIG. 46 is a graph illustrating VAN removal percentage (F?) of AHCNC-CHA (20 mg ml_‘1) at Co, VAN below 100 pg mL-1, determined by HPLC. The data within the dashed bracket shows that the equilibrium VAN concentration was lower than the detection limit of HPLC, thus R ~ 75% is likely underestimated.

[0113] Fl G. 47 is a graph illustrating the optical density (ODeoo) of bacteria (VSEfrn), cultured in the supernatant of VAN after incubation with AHCNC-CHA at varying periods. The concentration of all sorbents (AHCNC, CHA, or AHCNC-CHA) was fixed at 20 mg mL-1. CHA, AHCNC, and AHCNC-CHA control groups are VAN-free.

[0114] Fl G. 48 is a graph illustrating the optical density (ODeoo) of bacteria (VSEfrn), cultured in the supernatant of VAN after incubation with varying AHCNC-CHA doses for 4 h.

[0115] Fl G. 49 is a graph illustrating optical density (ODeoo) of bacteria (VSEfm), cultured in the supernatant of VAN incubated with varying acid (HCI, 5 M)-treated AHCNC-CHA doses for 4 h.

[0116] FIG. 50 is a graph illustrating the fecal density at each time point for total E. f aecium.

[0117] Fl G. 51 is a graph illustrating the fecal density at cumulative shedding for total E. faecium. Cumulative shedding is defined as the total area under the fecal density time-series curve (AUC) for the duration of the study. Data represent the mean ± SEM, and significant differences were determined by the Kruskal-Wallis test,Atty. Ref. No. 0073605-000956followed by the Dunn’s post-hoc test for pairwise multiple comparisons (*P< 0.05 and **P<0.01).

[0118] Fl G. 52 is a schematic illustration of a timeline and protocol of the mouse model used to assess the anti-VAN capability of AHCNC-CHA in vivo.

[0119] Fl G. 53 is a graph illustrating daily changes in body mass (% change relative to day 0) following the twice daily treatment with either 0, 4, or 10 mg AHCNC-CHA.

[0120] Fl G. 54 is a graph illustrating the fecal density at each time point for VRE / m.

[0121] FIG. 55 is a graph illustrating the fecal density at cumulative shedding for VRE / m. Cumulative shedding is defined as the total area under the fecal density timeseries curve (AUC) for the duration of the study. Data represent the mean ± SEM, and significant differences were determined by the Kruskal-Wallis test, followed by the Dunn’s post-hoc test for pairwise multiple comparisons (*P < 0.05 and **P < 0.01 ).

[0122] FIG. 56 is a graph illustrating the fecal density at each time point for VSE / m.

[0123] Fl G. 57 is a graph illustrating the fecal density at cumulative shedding for VSE / m. Cumulative shedding is defined as the total area under the fecal density timeseries curve (AUC) for the duration of the study. Data represent the mean ± SEM, and significant differences were determined by the Kruskal-Wallis test, followed by the Dunn’s post-hoc test for pairwise multiple comparisons (*P < 0.05 and **P < 0.01 ).

[0124] Fl G. 58 is a graph illustrating the proportion of VRE / m (of total E. faecium) at each time point.

[0125] Fl G. 59 is a graph illustrating the cumulative proportion of VRE / m (of total E. faecium) across all time points. Data represent the mean ± SEM, and significant differences were determined by the Kruskal-Wallis test, followed by the Dunn’s post-hoc test for pairwise multiple comparisons (*P< 0.05 and **P< 0.01).DETAILED DESCRIPTION OF THE INVENTIONAtty. Ref. No. 0073605-000956

[0126] 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.

[0127] Fl G. 1 provides an illustration of an exemplary embodiment of a process for the removal of one or more antibiotic compounds within a patient’s Gl tract while that patient is undergoing one or more intravenous (IV) antibiotic treatments via one or more intravenous injections. Exemplary antibiotic compounds may include, but not be limited to, B-lactams, aminoglycosides, chloramphenicol, glycopeptides, quinolones, oxazolidinones, sulfonamides, tetracyclines, macrolides, ansamycins, streptogramins, lipopeptides, or combinations thereof. An exemplary antibiotic compound may include, but not be limited to, vancomycin (VAN). In some embodiments, the IV antibiotic treatment can include VAN alone or VAN in combination with one or more other antibacterial compounds (e.g. linezolid, daptomycin (DAP), etc.). In other embodiments, the IV antibiotic treatment can include DAP and / or other antibacterial compounds (e.g. linezolid, etc.).

[0128] A patient can orally take one or more capsules, pills, or other type of orally configured receptacle that can include an antibiotic removal agent or an antibiotic deactivation agent. 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, such 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, be in 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 Gl tract via its oral digestion.Atty. Ref. No. 0073605-000956

[0129] As used herein, the term “patient” may refer to any biological system to which an antibiotic removal agent or an antibiotic deactivation agent can be administered, including without limitation, humans, other animals, (e.g., dogs, cats, horses, cows, pigs, chickens, cattle, etc.), pets, etc.

[0130] The oral digestion of the antibiotic removal agent or antibiotic deactivation agent can occur during the IV antibiotic treatment, before the IV antibiotic treatment, or shortly after the IV antibiotic treatment in different embodiments. The oral digestion of the antibiotic removal agent or antibiotic deactivation agent can alternatively occur during or shortly after an antibiotic compound 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.).

[0131] 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 Gl tract of a patient so that after an IV treatment of one or more antibiotic compounds, the antibiotic removal agent or antibiotic deactivation agent is present in the Gl tract for removal or deactivation of any of the one or more antibiotic compounds from the patient Gl tract so that any antibiotic compound(s) that may pass into the patient’s Gl tract after being injected into the patient can be effectively removed from the Gl tract or effectively deactivated from the Gl 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)).

[0132] In some embodiments, the patient can be on an oral digestion protocol for repeatedly taking the antibiotic removal agent or antibiotic deactivation agent 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 treatment 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.Atty. Ref. No. 0073605-000956

[0133] As can be appreciated from the left side of the schematic illustration of FIG. 1, the antibiotic removal agent or antibiotic deactivation agent can be configured to react with excess antibiotic compounds (e.g., VAN) present in the patient Gl tract after the patient is injected with the antibiotic compound(s). The removal can occur via the antibiotic compounds being adsorbed into the antibiotic removal agent or antibiotic deactivation agent due to an affinity the antibiotic removal agent can have for the antibiotic compound(s). The removal may also occur by a chemical reaction or other type of interaction with the antibiotic removal agent or antibiotic deactivation agent and the antibiotic compound(s) in the Gl 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 or antibiotic deactivation agent in the patient Gl tract.

[0134] As can be appreciated from a comparison of the right side of FIG. 1 with the left side of FIG. 1 , the use of the antibiotic removal agent or antibiotic deactivation agent can avoid antibiotic compounds being bioreactive in the patient’s Gl tract so that those compounds do not kill 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 Gl tract while that patient undergoes an IV antibiotic treatment or other type of antibiotic treatment.

[0135] Fl G. 12 provides an illustration of an exemplary antibiotic removal agent or antibiotic deactivation agent. In exemplary embodiments, the antibiotic removal agent or antibiotic deactivation agent includes hairy cellulose nanocrystals (HCNC) hybridized with, or immobilized on, a resin. In other embodiments, the antibiotic removal agent or antibiotic deactivation agent includes functionalized biopolymers. In still other embodiments, the antibiotic removal agent or antibiotic deactivation agent includes hairy cellulose nanocrystals (HCNC) hybridized with, or immobilized on, a resin, in combination with functionalized biopolymers.

[0136] The HCNC may include cellulose (polysaccharide) bodies, such as cellulose fibrils and / or crystals. In some embodiments, the cellulose bodies may beAtty. Ref. No. 0073605-000956nanoscale bodies. For example, the cellulose bodies may be nanofibrils and / or nanocrystals. The cellulose bodies may be derived from any suitable source. In some embodiments, the cellulose bodies may be biosourced (e.g., derived from biomass). For example, the cellulose bodies may be plant-based, such as derived from wood pulp, cotton, algae, or any other suitable plant-base source. In some embodiments, the cellulose bodies may be non-toxic and and / or biodegradable.

[0137] The cellulose bodies may have a plurality of cellulose chains (also referred to as “hairs”) protruding therefrom, such as protruding from the ends of the bodies. The cellulose chains may be functionalized with anionic groups. The anionic groups may be selected from the group consisting of carboxylates, dicarboxylates, sulfates, or any other suitable anionic group. The anionic groups may serve as active sites configured to adsorb to the antibiotic compound(s).

[0138] In some embodiments, the cellulose chains are functionalized with cationic groups. In some embodiments, the cellulose chains are functionalized with amphiphilic groups. In some embodiments, the cellulose chains are functionalized with zwitterionic groups. In some embodiments, the cellulose chains are functionalized with a combination of electrically neutral anionic, cationic, zwitterionic, and / or amphiphilic groups.

[0139] The cellulose bodies may be any shape and / or size, though it is contemplated that the bodies may be rod-like or needle-like structures. Advantageously, the cellulose bodies are elongated in shape; that is, having a length / width ratio higher than 1.

[0140] The HCNC may be hybridized with, or immobilized on, a resin. The resin may be in particulate form, such as in microparticulate form. In particular, the HCNC may be adsorbed to the surface of the particulate resin and / or within the pores of the particulate resin. The resin may therefore advantageously be porous.

[0141] In some embodiments, the particulate resin may include cholestyramine (CHA), patiromer, sodium polystyrene sulfonate, sodium zirconium cyclosilicate, sodium polystyrene sulfonate, sodium polystyrene sulfonate, colestipol, tolevamer, polacrilexAtty. Ref. No. 0073605-000956resin (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.

[0142] In some embodiments, the resin includes or consists of cholestyramine (CHA). In such embodiments, the antibiotic removal agent or antibiotic deactivation agent may be designated as an HCNC-CHA agent.

[0143] The functionalized biopolymers may include polysaccharide, protein, polynucleotides, and / or polypeptides, wherein the biopolymer is functionalized with a combination of electrically neutral anionic, cationic, zwitterionic, and / or amphiphilic groups.

[0144] As can be appreciated from the above, embodiments generally relate to processes and apparatus configured to reduce, hinder, or avoid the growth or development of drug resistant bacteria and / or the protection of drug sensitive bacteria. Embodiments can additionally, or alternatively, be adapted to help block or avoid antibiotic-resistant bacterial growth within the body of a patient, for example.Embodiments can additionally, or alternatively, be adapted to help protect or promote antibiotic-sensitive bacterial growth within the body of a patient, for example.Embodiments can additionally, or alternatively, be adapted to help protect the microbiome within the body of a patient, for example.

[0145] Fl G. 12 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 Gl tract, for example. The antibiotic removal agent or antibiotic deactivation agent can render the antibiotic compounds inert to the Gl 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 or the antibiotic deactivation agent particulate. The antibiotic removal agent or antibiotic deactivation agentAtty. Ref. No. 0073605-000956consumed 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.

[0146] Embodiments of the 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 Gl tract.

[0147] In some embodiments, the one or more antibiotic compounds may be used to treat an infectious disease in a patient. The infectious disease may be located in the Gl tract, or may not be located in the Gl tract. In other embodiments, the one or more antibiotic compounds may be used to treat a non-infectious disease in a patient. The non-infectious disease may be located in the Gl tract, or may not be located in the Gl tract.

[0148] 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 Gl tract and / or have VAN’s bioreactivity with bacteria removed so that the removed VAN does not affect the patient’s Gl tract bacteria (e.g., it can avoid the Gl tract bacteria from dying due to the antibacterial nature of the VAN after adsorption and / or absorption by the antibiotic removal agent). For example, the agents, when ingested by the patient, can function to block the activity of VAN (or other antibiotic compound(s)) at physiologically relevant concentrations to effectively remove the antibiotic compounds from the patient’s Gl 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 antibiotic compound(s) in the patientAtty. Ref. No. 0073605-000956Gl tract via the antibiotic removal agent having or being one or more antibacterial agent removal compounds or antibacterial agent deactivation compounds in some embodiments.

[0149] For example, as discussed below, we conducted HCNC-CHA agent VAN removal via conducting in vitro experiments at controlled initial antibiotic concentrations, pH, and ionic strengths. The mechanisms of antibiotic removal by HCNC-CHA 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).EXAMPLESMethods and Materials

[0150] In our conducted evaluation and experimentation work, Northern bleached softwood sheets (Domtar Inc., Canada) served as the starting cellulose material.Sodium (meta)-periodate (NaIC , > 99.0%), sodium chloride (NaCI, > 99.5%), sodium chlorite (NaCIC>2, 80%), hydrogen peroxide (H2O2, 30 wt%), sodium hydroxide (NaOH, ACS Reagent, > 97%), hydrochloric acid (HCI, ACS reagent, 37%), ethylene glycol (Reagent plus > 99%), poly-L-lysine (PLL, 0.1% w / v), hydroxylamine hydrochloride (NH2OH HCI, Reagent plus 99%), calcium chloride dihydrate (CaCl2-2H2O, for molecular biology, > 99.0%), centrifuge tube filter cellulose acetate membrane, 0.22 pm pore size), L-a- phosphatidylcholine (type XVI-E, > 99.0% ), and cholestyramine resin (CHA, Dowex® 1x2 chloride form) were purchased from MilliporeSigma (USA). BA (C26H44NNaO?S) and MA (C4H4O4, > 98%) were purchased from Spectrum Chemical and Beantown Chemical (USA), respectively. Mica sheets (V1 grade) and Krazy glue were purchased from Ted Pella Inc. (USA) and Agar Scientific (UK), respectively.Fasted state simulated intestinal fluid (FaSSIF-V2) and fasted state simulated gastric fluid (FaSSGF) in the powder form were procured from Biorelevant (UK). Vancomycin hydrochloride (USP grade, > 95%) was purchased from VWR International (USA).Anhydrous ethanol (200 proof) was supplied by KOPTEC (USA). Conductive doubleAtty. Ref. No. 0073605-000956sided adhesive carbon tapes were purchased from Rave Scientific (USA). Formic acid (CH2O2, > 99%), Optima™ water (liquid chromatography-mass spectroscopy (LC-MS) grade), and acetonitrile (Honeywell Burdick & Jackson Brand™, C2H3N, > 99.9%) were procured from Thermo Fisher Scientific (USA). Milli-Q water (resistivity = 18.2 mQ cm at 25 °C) was generated from deionized (DI) water using a remote water purification system (Direct-Q 5, Millipore, USA).

[0151] The bacterial culture media, Mueller Hinton II Broth (cation-adjusted), Enterococcosel Agar (BBL formulation), and Brain Heart Infusion Broth (BHI, BBL formulation were purchased from BD Difco (USA). Two E. faecium clones, BL00239-1, and BL00239-1S, were derived from a clinical isolate collected from a patient undergoing treatment for a VRE / m bloodstream infection at the University of Michigan hospital. Phosphate-buffered saline (PBS) solution (1 X) was purchased from Thermo Fisher Scientific (USA). Glycerol was obtained from VWR International (Molecular biology grade, USA). Sterile 0.9% saline solution was supplied from Aspen Veterinary Solutions (USA). Eight-week-old female Swiss-Webster mice were purchased from Charles River (USA), and 5053-PicoLab Rodent Diet 20 was supplied by LabDiet (USA). Veterinary grade VAN for mouse injection and ampicillin were obtained from Slate Run Pharmaceuticals (USA) and Alfa-Aesar, respectively.

[0152] AHCNC synthesis: Pulp sheets (1 g) torn into ~ 2 x 2 cm2thin pieces were soaked in DI water. The wet pulp disintegrated via mechanical stirring overnight and then vacuum-filtered. Afterwards, NaCI (3.8 g) and NalC>4 (1.32 g) were dissolved in DI water (total volume = 65 mL, including the remaining water in the wet pulp) in an aluminum foil-wrapped beaker to prevent light-triggered NalCM deactivation, followed by the addition of wet pulp and stirring at ambient temperature for 42 h.

[0153] To stop the oxidation reaction by quenching the unreacted NaIC , 1 mL of ethylene glycol was added to the mixture. Partially oxidized fibrils, called dialdehyde-modified cellulose (DAMC), were rinsed at least 5 times with DI water, followed by vacuum filtration. Never-dried DAMC was then oxidized via reacting with NaCIC (0.8452 g) and H2O2 (0.8478 mL) in DI water (50 mL). NaOH (0.5 M) was intermittentlyAtty. Ref. No. 0073605-000956added during reaction while stirring the mixture for 12 h at room temperature to maintain the pH at 5. After completion, the mixture was centrifuged at 27000 *g for 15 min to eliminate any residual unfibrillated fibrils. The AHCNC was separated from the collected supernatant via precipitation using ethanol as a non-solvent (0.16 g per 1 g of dispersion). The milky dispersion was then centrifuged at 3000 xgfor 15 min. Finally, excess ethanol was added to the remaining supernatant to precipitate fully solubilized dicarboxylated cellulose (DCC) polymers, separated by centrifugation at 3000 xg. The AHCNC and DCC were redispersed separately in DI water (~50 mL) and dialyzed (Spectra / Por dialysis bags, molecular weight cutoff = 6 - 8 kDa) against DI water for 3 days, followed by concentration measurement via oven drying and weighing 1 mL of dispersion.

[0154] AHCNC-CHA hybridization: AHCNC-CHA biomaterials were prepared by incubating CHA microparticles (20 mg) with varying concentrations of AHCNC dispersions (total volume = 2 mL) for 24 h at ambient temperature. The mixture was then centrifugated at 15000 xg for 5 min, the supernatant was removed, and Milli-Q water (1 mL) was used to rinse the samples at least 3 times to remove any unbound AHCNC. The quantity of AHCNC immobilized onto CHA was measured by subtracting the dry mass of free AHCNC in the supernatant from the total mass of initial AHCNC. The AHCNC-CHA was then dried in the oven at 37°C overnight for further experiments.

[0155] Aldehyde group content measurement: The aldehyde group content of DAMC, the intermediate product for AHCNC synthesis, was quantified using an oxime titration method. A certain amount of wet DAMC fibrils was suspended in Milli-Q water (50 mL), and the pH was adjusted to 3.5 by adding HCI (0.1 M), followed by adding a NH2OH HCI solution (5 wt%, 10 mL) with the same pH (3.5, adjusted using a 0.1 M NaOH solution) and stirring. The reaction of aldehyde groups with NH2OH-HCI (oxime reaction) yielded HCI. The pH decrease was compensated via gradually adding a NaOH solution (10 mM, 40 mL) at a rate of 0.1 mL min-1using an automatic titrator (Metrohm 907 Titrando, USA). The amount of NaOH consumed to reach the initial pH (i.e., 3.5) was used to calculate the DAMC aldehyde content.Atty. Ref. No. 0073605-000956

[0156] Carboxylate group content measurement: Carboxylate group content of AHCNC before and after immobilization onto the CHA resin was quantified by conductometric titration. A certain amount of material (20 mg) was dispersed / suspended in Milli-Q water (140 mL), including 2 mL of NaCI solution (20 mM). Then, pH was reduced to 3 via adding a HCI solution (stock solution concentration = 0.1 M). Using the automatic titrator, the mixture was titrated with a NaOH solution (10 mM) at a rate of 0.1 mL min-1until the pH reached ~ 11. The content of carboxyl groups was obtained based on the middle portion of titration curves, associated with the weak acid.

[0157] Quantification of adsorbed AHCNC in the AHCNC-CHA: To quantify adsorbed AHCNC in AHCNC-CHA, the suspension of AHCNC-CHA was centrifugated at 15000 xg for 5 min after hybridization. Then, the supernatant (1 mL) was dried at 37 °C in an oven overnight to measure the free (unadsorbed) AHCNC mass ( / 7?AHCNC). The AHCNC adsorption ratio (mAAHCNc / mcHA) was then calculated using Equation (1):>

[0158] where m0 AHCNc represents the initial mass of AHCNC before adding to the CHA suspension, and mCHA(mg) denotes CHA mass in the AHCNC-CHA biomaterial.

[0159] To quantify the desorbed AHCNC from AHCNC-CHA, the AHCNC-CHA suspension was incubated at varying pH for 6 h, followed by centrifugation at 15000 xg for 5 min. Then, the supernatants were dried at 37°C in an oven overnight to calculate the desorbed AHCNC mass (mo, AHCNC). The AHCNC desorption ratio ( / T?D,AHCNC / / 7?AHCNC-CHA) was then measured via dividing mo, AHCNC by the initial mass of AHCNC-CHA (mAHCNc-GHA) incubated at varying pH.

[0160] Atomic force microscopy (AFM) imaging: To investigate the morphology of AHCNC, a multimode AFM (Broker Dimension Icon, USA) was used in the PeakForce tapping mode using a silicon nitride probe (Broker ScanAsyst-Air, USA). To prepare the AHCNC sample for imaging, freshly cleaved mica was adhered to a stainless-steel disc using the Krazy glue and pre-coated with a PLL solution (10 pL, 0.1% w / v). After 10 min, the excess PLL was rinsed with Milli-Q-water. Then, 10 pL of AHCNC dispersion (0.1 mg mL-1) was placed on the mica sheet, air-dried overnight,Atty. Ref. No. 0073605-000956rinsed gently 5 time with 100 pL of Milli-Q water, and air-dried overnight again before AFM imaging. The AFM images were analyzed using the NanoScope Analysis software (Version 1.80, accessed via Penn State Materials Characterization Laboratory, MCL). Length and width of > 50 particles per image (n = 3) were measured by the Gwyddion software (Version 2.49, accessed via Penn State MCL).

[0161] Hydrodynamic size and potential assessments: The hydrodynamic equivalent size of AHCNC, VAN, and AHCNC-VAN was measured at 25 °C and 90° scattering angle using a dynamic light scattering (DLS) instrument (Malvern Zetasizer Nano series, UK). Before conducting the measurements, the samples were diluted to 0.1% w / v using ultrapure water at desired pH (adjusted using a 2M NaOH or 1M HCI solutions), Na+concentration, and / or Ca2+concentrations. Additionally, the hydrodynamic size of AHCNC (0.1% w / v) was measured at varying initial VAN concentrations (0-10 mg mL-1, pH = 6.5, adjusted using 0.5 M NaOH). Subsequently, the samples (70 pL) were pipetted into low-volume quartz cuvettes (ZEN2112, Malvern, UK) to conduct the spectroscopy. The Z-average (cumulants mean) of intensity measurements were reported as the hydrodynamic equivalent size of particles.

[0162] The ^-potential of AHCNC, VAN, and AHCNC-VAN was estimated based on the electrophoretic mobility, measured by the Nano ZS Zetasizer (Malvern instrument, UK). Before measuring ^-potential, samples were diluted to 0.1% w / v using ultrapure water at desired pH (adjusted using a 2 M NaOH or 1 M HCI solutions), Na+concentration, and / or Ca2+concentrations. Additionally, the ^-potential of AHCNC (0.1% w / v) was measured at varying initial VAN concentrations (0-10 mg mL-1, pH = 6.5, adjusted using 0.5 M NaOH). Samples (900 pL) were then pipetted into the universal dip cell kit (Malvern, UK) for conducting the measurements. The particles have an average width of ~ 7 nm, and f (Ka) ~ 0.5 (K and a denote the Debye-Huckel parameter and the particle radius, respectively). Accordingly, the ^-potential of particles was estimated by applying Equation (2) to the electrophoretic mobility.<Atty. Ref. No. 0073605-000956

[0163] where / j\\ and ± denote the electrophoretic mobility for cylindrical particles in the parallel and perpendicular direction to the electric field, respectively, is the relative dielectric permittivity of solvent, co denotes the dielectric permittivity in a vacuum i.e., vacuum permittivity, and rj is the viscosity. For VAN, ^-potential was estimated using the Smoluchowski equation.

[0164] Attenuated total reflectance (ATR)-Fourier transform infrared (FTIR) spectroscopy: The functional groups of AHCNC, CHA, and AHCNC-CHA were characterized using a Vertex 70 FTIR spectrometer (Bruker, USA) with a Harrick Diamax ATR accessory following the principles of the Bouguer-Beer-Lambert law. The samples were dried at 37 °C for two days to ensure water removal, eliminating the water absorption peaks. The dried samples were placed onto the ATR crystal, and the tip of pressure clamp was gently lowered to apply maximum pressure. The ATR-FTIR spectra were obtained by averaging a total of 100 scans in a wavenumber range of 500 - 4000 cm-1(resolution = 6 cm-1). The spectrum of the bare diamond was used as a reference to establish a baseline and normalize the absorbance values.

[0165] Scanning electron microscopy (SEM) imaging: Dried CHA or AHCNC-CHA were secured to sample pins using a double-sided adhesive carbon tape and coated with a 3 nm-thick indium layer using a vacuum sputtering instrument (Leica sputter coating EM ACE 600, USA). The particles were then imaged under SEM (Thermo Scientific Verios G4, USA) with an accelerating voltage of 3.0 kV and a beam current of 0.1 nA.

[0166] Surface area and pore size analyses: Nitrogen adsorption and desorption isotherm measurements were conducted on a Micromeritics instrument (ASAP™ 2420, Micromeritics Instrument, GA, USA) at 77 K. CHA and AHCNC-CHA samples were degassed under vacuum for 4 h prior to measurements to remove any unwanted vapors and gases, and then transferred to analysis ports. The specific surface area was determined using Brunauer-Emmet-Teller (BET) equation.Additionally, pore volume and pore diameter were obtained using the Barrett-Joyner-Atty. Ref. No. 0073605-000956Hollande (BJH) model. The calculations were performed using the ASAP™ 2420 software (package V6.07).

[0167] In vitro VAN removal: In all the removal experiments, three steps were followed: (i) VAN stock solutions (concentration = 40 mg mL’1) were prepared by dissolving 800 mg of VAN in 20 mL of Milli-Q water. To prepare the VAN solutions with desired concentrations, the stock solution was further diluted using Milli-Q water, (ii) standard calibration lines were generated for each experimental condition by recording the absorbance of known concentrations of VAN solutions, ranging from 0.05 mg mL-1to 10 mg mL-1at the maximum peak of wavelength (Amax) = 280 nm. A total of 12 calibration lines were used to calculate VAN concentration in the supernatants, analyzed by an UV-vis spectrophotometry (Tecan Model Infinite 200 Pro, USA), (iii) The VAN removal percentage (R, %) and the equilibrium VAN removal capacity (qe, mg g-1) were calculated according to Equations (3) and (4), respectively:

[0168] where Co (mg mL’1) and Ce(mg mL’1) are the initial and equilibrium VAN concentrations, respectively, m (g) is the mass of sorbent material, and V (mL) is the total volume of solution. Note that the detection limit of VAN concentration by UV-vis spectrophotometry was -0.05 mg mL’1. The measured equilibrium VAN concentrations (i.e. Ce) lower than the detection limit were all assumed as 0.05 mg mL’1. Therefore, the Rvalues calculated via applying this assumption are all likely to be underestimated.

[0169] Effect of initial VAN concentration on VAN removal: The capability of AHCNC or AHCNC-CHA in removing VAN was investigated via batch removal experiments. VAN solutions were prepared by diluting the VAN stock solution with Milli-Q water to obtain varying concentrations (1-20 mg mL’1). The pH of all solutions was adjusted to 6.5 by the addition of NaOH solution (0.5 M) and Milli-Q water to a total desired volume of 1 mL. The AHCNC (final concentration = 0.25 mg) or AHCNC-CHA (20 mg) were added to the VAN solutions (final volume = 1 mL), vortexed for 1 min, and the vials were maintained on the nutating mixer (Fisherbrand, USA) to shake at 60 rpmAtty. Ref. No. 0073605-000956at room temperature. Agitation time varied depending on the experiment type. After centrifugation at 5000 x for 5 min, supernatants were collected, and the VAN concentration was determined using the UV-vis spectrophotometer at Amax of 280 nm.

[0170] Effect of contact time on VAN removal: To investigate the effect of contact time on AHCNC or AHCNC-CHA-mediated VAN removal, VAN solutions with a constant concentration (1 mg mL-1, total volume = 1 mL, including the volume of acid or base used for pH adjustment to 6.5) were prepared by diluting VAN stock solutions with Milli-Q-water. The pH of all solutions was increased to 6.5 by adding a NaOH solution (0.5 M). AHCNC-CHA (20 mg) was then added to the VAN solutions, vortexed for 1 min, and the vials were placed on the nutating mixer, set to 60 rpm. At specific time intervals (5 min, 1, 2, 4, and 24 h), the samples were centrifuged at 5000 xg for 5 min. Then, the supernatants were separated, and the VAN concentration was analyzed using the UV-vis spectrophotometer at Amax = 280 nm.

[0171] Effect of AHCNC-CHA dose on VAN removal: To examine the effect of AHCNC-CHA dose on VAN removal, VAN solutions with a concentration of 1 mg mL-1were prepared by diluting the VAN stock solution and adjusting the pH to 6.5 using a NaOH solution (0.5 M). Varying concentrations of AHCNC-CHA were then added to the VAN solutions, followed by vortexing for 1 min and maintaining the vials on the nutating mixer to agitate at 60 rpm. After 4 h of incubation, supernatants were separated by centrifugation at 5000 xg for 5 min and analyzed by a UV-vis spectrophotometer at Amax— 280 nm.

[0172] Effect of pH on VAN removal: To investigate the ionization effects of AHCNC-CHA and VAN functional groups on VAN removal, the experiments were performed in VAN solutions at pH ranging from 2 to 12. VAN solutions with a constant concentration (1 mg mL-1) were prepared by diluting the VAN stock solution in vials and adjusting the pH by adding a NaOH or HCI solution and Milli-Q water to a total solution volume of 1 mL. Then, AHCNC-CHA (20 mg) was added to the VAN solutions with varying pH, followed by vortexing for 1 min and maintaining the vials on the nutatingAtty. Ref. No. 0073605-000956mixer at 60 rpm. After 4 h of incubation, supernatants were separated by centrifugation at 5000 xg for 5 min and analyzed by a UV-vis spectrophotometer at Amax= 280 nm.

[0173] Effect of ionic strength and ion type on VAN removal: To explore the effect of ion types and ionic strength on AHCNC-CHA-mediated VAN removal, the removal experiments were conducted at varying Na+or Ca2+concentrations ranging from 0 to 200 mM. VAN solutions with a concentration of 1 mg mL-1were prepared by diluting the VAN stock solution with Milli-Q water and adjusting the pH to 6.5 using a NaOH solution (0.5 M). AHCNC-CHA (20 mg) was then added to the VAN solutions containing varying Na+or Ca2+concentrations and vortexed for 1 min. The vials were maintained on the nutating mixer while agitating at 60 rpm. After 4 h of incubation, supernatants were separated by centrifugation at 5000xg for 5 min and analyzed using the UV-vis spectrophotometer at Amax= 280 nm.

[0174] Preparation of simulated intestinal fluid (SIF) and simulated gastric fluid (SGF) media: To simulate SIF in vitro, fasted state SIF (FaSSIF) and fed state SIF (FeSSIF) were formulated according to the standard protocols. For the FaSSIF, NaOH pellets (1.392 g), MA (2.22 g), and NaCI (4.01 g) were first dissolved in Milli-Q water (0.9 L). The pH of the solution was raised to 6.5 by adding a NaOH solution (0.1 M), and the total volume was adjusted to 1 L using Milli-Q water. Then, FaSSIF-V2 powder (1.79 g) was added to the solution and allowed to stir for 1 h at the ambient temperature. For the FeSSIF, sodium taurocholate (8.25 g) was added to the FaSSIF (250 mL) and stirred at the ambient temperature to fully dissolve. Subsequently, PC (2.95 g) was added to the solution and the final volume was adjusted to 1 L using FaSSIF. Finally, the mixture was stirred for an additional 4 h until it became clear.

[0175] To prepare the SGF solution, NaCI (1.999 g) and HCI (29.1 g, 1 M) were dissolved in Milli-Q water (0.9 L). The pH of solution was maintained at 1.6 using a HCI solution (1 M), and the total volume was adjusted to 1 L using Milli-Q water. Then, FaSSGF powder (0.06 g) was added to the solution and stirred for 1 h at the ambient temperature.Atty. Ref. No. 0073605-000956

[0176] Effect of AHCNC-CHA pre-incubation time in SGF on VAN removal:AHCNC-CHA (100 mg) was first incubated in the SGF (5 mL) for varying periods (1-6 h) at 37 °C to mimic the food retention in the stomach. Afterwards, the suspensions were centrifugated at 15000 xg for 5 min, the supernatants were decanted, and Milli-Q water was used to wash away any remaining SGF. The precipitates, SGF-incubated AHCNC-CHA, were then oven-dried at 37 °C overnight. To determine the effect of SGF incubation time on AHCNC-CHA-mediated VAN removal, SGF-incubated AHCNC-CHA (20 mg) were added to the VAN solutions (final concentration = 1 mg mL-1, pH = 6.5, total volume = 1 mL). The vials were then placed on the nutating mixer, agitating at 60 rpm. After 4 h of incubation, supernatants were separated by centrifugation at 5000 xg for 5 min and analyzed by the UV-vis spectrophotometer at Amax= 280 nm.

[0177] Effect of SIF components on VAN removal: To examine the effect of SIF components on VAN removal, AHCNC-CHA (20 mg) was added to the VAN solutions (final concentration = 1 mg mL-1, total volume = 1 mL, pH = 6.5) containing varying concentrations of BA (1-10 mM), MA (10-100 mM) or PC (1-4 mM). The vials were then maintained on the nutating mixer at 60 rpm for 4 h, followed by separating the supernatants by centrifugation at 5000 xg for 5 min and analyzing using the UV-vis spectrophotometer at Amax— 280 nm.

[0178] Effect of SIF on VAN removal: To investigate the effect of SIF on VAN removal, VAN solutions with varying concentrations (1-20 mg mL-1) were prepared by diluting the VAN stock solutions with either FaSSIF or FeSSIF. The final volume of VAN solutions was 1 mL after the pH adjustment to 6.5 using a NaOH solution (0.5 M).AHCNC-CHA (20 mg) was then added to the VAN solutions, agitated at 60 rpm using the nutating mixer for 4 h. The supernatants were then separated by centrifugation at 5000 xg for 5 min and analyzed by the UV-vis spectrophotometer at Amax= 280 nm.

[0179] High-performance liquid chromatography (HPLC): An HPLC instrument (Shimadzu LC-20AD UFLC XR, Japan), equipped with a UV detector was used to measure VAN concentrations below 100 mg L“1. The mobile phase consisted of 0.1% v / v formic acid in Optima™ liquid chromatography-mass spectroscopy (LC-MS)Atty. Ref. No. 0073605-000956grade water and formic acid in acetonitrile (0.1% v / v). Chromatographic separation was conducted using an Acuity UPLC BEH C18 analytical column (length: 150 mm, inner diameter: 2.1 mm, particle size: 1.4 m) (Acuity, USA). The injection volume and the flow rate were 1 pL and 0.25 mL min-1, respectively. The column temperature was maintained at 55 °C. The peak areas detected at 205 nm were defined as analytical signs.

[0180] Uncaptured VAN antibiotic activity via a broth microdilution assay assay: Broth microdilution assays were used to quantify the effects of AHCNC-CHA on the antibiotic activity of VAN against VSEfm. VAN removal was conducted in Milli-Q water at pH = 6.5 using varying AHCNC-CHA doses and incubation time. After centrifugation at 16,300xg for 5 min, the supernatant of each sample was separated and transferred to 0.22 pm cellulose acetate tube filters. All assays were performed according to the guidelines of the Clinical Laboratory Standards Institute using a patient-derived VSEfm (minimum inhibitory concentration MIC = 0.5 pg mL-1). Twofold serial VAN dilutions were prepared using the initial VAN concentrations (i.e., the VAN concentrations before AHCNC-CHA-mediated capture). The bacterial cell density was quantified after 24 h of incubation at 35 °C using a plate reader (BioTek Synergy H1 Plate reader, Agilent Technology, USA). The optical density was recorded at a wavelength of 600 nm, referred to as ODeoo. The data were fitted to a Hill function as previously described, with decreases in initial VAN concentrations leading to a right-shift in the bacterial growth curve.

[0181] Assessing the antibiotic activity of uncaptured VAN in vivo. The Pennsylvania State University Institutional Animal Care and Use Committee (PSU-IACUC, 47581) approved all animal procedures. Two previously described VREfm and VSEfrn isolates were used in all experiments. VREfrn clone BL00239-1 (MIC > 64.0 pg mL-1), expressing the vanA VAN resistance gene, was isolated from a patient bloodstream at the University of Michigan Hospital. \ / SEfm clone, BL00239-1S (Computed MIC = 1.0 pg mL-1, 95% Confidence interval - 0.8-1.3 pg mL-1) was derived through the in vivo mouse passage of BL000239-1 as previously described, with theAtty. Ref. No. 0073605-000956loss of vanA gene expression confirmed by polymerase chain reaction (PCR). Adult, 8-week-old female outbred Swiss Webster mice were used for this study. Given the absence of data suggesting sex-differences in VAN biliary excretion, only female mice were used to minimize animal use in accordance with PSU-IACUC guidelines. Mouse sample sizes (n = 5) were determined using Biomath (version 03 / 03) at a power of 0.8 and a 0.05 probability of type 1 error based on prior work with similar compounds. Mice were allowed to acclimatize to the on-site facility for at least 2 weeks under Biosafety Level (BSL)-2 housing conditions at 5 animals per microisolator cage prior to E. faecium inoculation and moved to individual cages thereafter. Sterile gowns, shoe covers, masks, eye protection, surgical bouffant, and double gloves were required for room entry. To prevent cross contamination when handling mice, outer gloves were changed, and all surfaces were thoroughly cleaned with 70% ethanol between cages. Mice were given irradiated feed (i.e., PicoLab Rodent Diet 20) and sterile water ad libitum.

[0182] To disrupt the intestinal microbiome and facilitate intestinal E. faecium colonization, mice were treated for 7 days with ampicillin (ad libitum; 0.5 g L-1in drinking water) prior to bacterial inoculation. VSEfm and VREfrn inoculations were grown overnight in BHI broth at 35 °C. Mice were subsequently inoculated via oral gavage with sterile saline (100 pL), containing approximately 108colony forming units (CFUs) at an approximately 1 : 20 ratio of VRE / TT? (BL00239-1) to VSE / m (BL00239-1S). Serial dilution on Enterococcosel agar plates was used to confirm inoculation density (1.3 x 108CFUs) and composition (6% VREfm). Beginning the day of E. faecium inoculation, mice were treated twice daily via oral gavage with either sterile saline (100 pL), sterile saline (100 pL) + AHCHC-CHA (4 mg), or sterile saline (100 pL) + AHCNC-CHA (10 mg) for 8 days. Beginning 1 day post inoculation, mice received veterinary grade VAN (30 mg kg-1) diluted in saline (0.9% w / v) via subcutaneous injection once daily for 5 days. Body mass was recorded daily for each mouse to monitor the safety and tolerability of the AHCNC-CHA compound, and fecal samples were collected at preselected time-points by placing mice in fresh unsealed plastic containers until defecation. Fecal pellets were subsequently transferred to pre-weighed 1.5 mL tubes byAtty. Ref. No. 0073605-000956autoclaved sterile toothpick, and fecal samples were dissolved in PBS (25 pL PBS mg-1feces) prior to freezing at -80 °C in glycerol (25%) until analysis by plating.

[0183] Total fecal E. faecium concentrations were quantified by serially diluting the previously frozen samples tenfold in PBS before plating on Enterococcosel agar and incubation for 40-48 h under aerobic conditions at 35 °C. E. faecium were identified by the ability to grow on Enterococcosel agar and colony morphology with each colony assumed to represent a single viable bacterium (CFU; colony forming unit) in the original sample. CFU counts were back calculated against the corresponding dilution factor to determine the total E. faecium concentrations in the initial fecal samples. Total CFU counts were normalized to CFUs per 10 mg of feces, and the limit of detection for the total E. faecium calculation was 5 CFUs per 10 mg of feces.

[0184] To enumerate fecal VREfm density, the calculated total E. faecium densities were used to plate 200 CFUs in triplicate on selective agar plates, supplemented with or without VAN (16 pg mL-1) for a total of 6 plates for each sample. Plates were then incubated for 40-48 h at 35 °C, and the difference in CFU counts between plates with or without VAN were used to quantify the proportion of VRE / m and VSEfm in each sample. Cumulative total E. faecium, VREfrn, and VSEfrn shedding was calculated from the area under the fecal density time series (Area Under the Curve: AUC) for the duration of the study (14 days).

[0185] Statistical Analyses: All in vitro data were collected from at least three independent experiments and reported as mean ± standard deviation (SD). All in vivo data represent the mean ± standard error of the mean (SEM). Significant differences between mean values were determined by the non-parametric Kruskal-Wallis test, followed by Dunn’s post-hoc test for pairwise multiple comparisons. Statistical analyses were performed using GraphPad Prism software (version 10.0.3), and mean differences were considered significant at p-values < 0.05. The symbols *, **, ***, and **** represent p-values of p < 0.05, p £ 0.01 , p £ 0.001 , and p £ 0.0001 , respectively. The p-values of p30.05 are considered non-significant.ResultsAtty. Ref. No. 0073605-000956

[0186] Design of AHCNC-CHA anti-antibiotics: Our process is based on the oral administration of AHCNC-CHA sorbent for Gl-exposed VAN removal. To implement this approach, we first engineered highly negatively charged AHCNC, providing active sites for VAN removal, and hybridized it with CHA, yielding the AHCNC-CHA sorbents. FIG. 2 shows the synthesis of AHCNC from softwood cellulose pulp fibrils through sequential oxidation reactions. The first 42 h periodate reaction yielded intact DAMC fibrils, which were further converted to a mixture of dicarboxylate-bearing AHCNC and DCC via a 12 h chlorite oxidation reaction. AHCNC were then isolated by nonsolvent-mediated precipitation.

[0187] FIG. 3 presents the needle-like crystalline body of AHCNC, obtained by AFM imaging. The length and width of AHCNC were 96 + 26 nm and 7 ± 2 nm, respectively, i.e. , within the same order of magnitude of conventional (strong-acid hydrolyzed, non-hairy) CNCs. It has been indirectly shown that AHCNC comprise a crystalline body with polyanionic disordered cellulose chains protruding from both ends. The aldehyde content of DAMC, an intermediate product for the AHCNC synthesis, was determined by measuring the NaOH volume required to neutralize HCI released as the aldehyde groups reacted with hydroxylamine-hydrochloride. The aldehyde content was 6.0 ± 0.3 mmol g-1(an example shown in FIG. 4), which were then converted to carboxylate groups to disintegrate DAMC and form AHCNC. The carboxylate content of AHCNC was 6.0 ± 0.4 mmol g-1, which was assessed by the conductometric titration (an example shown in FIG. 5).

[0188] AHCNC, bearing a high content of polyanionic groups, were used as the active anti-antibiotic component. Assuming the adsorption of VAN to AHCNC is electrostatically driven, 5.57 mmol of carboxylate groups per 1 g of AHCNC adsorbs an equal molar amount of amine groups on VAN (theoretical amine group content of VAN is 1.34 mmol g-1); thus, 0.25 mg mL-1of AHCNC stoichiometrically binds 1.11 mg mL-1of VAN. To investigate the AHCNC-mediated VAN removal kinetics, FIG. 6 shows the VAN removal percentage ( ?) and capacity (qe) of AHCNC as a function of incubation time. AHCNC (0.25 mg mL-1) were incubated with a supra-stochiometric VANAtty. Ref. No. 0073605-000956concentration (6 mg mL-1, 1 mL) to identify the effect of contact time on VAN removal. In this condition, R - 30% and qe~ 7700 ± 1280 mg of VAN per 1 g of AHCNC was achieved in less than 10 s. The qeobtained here was - 2 times higher than the stoichiometric value, 4038 ± 290 mg g-1, based on the charge stoichiometry. The higher experimental qemay imply that the electrostatic interactions of only one VAN amine group (out of two per molecule) with one carboxylate group of AHCNC may be sufficient for AHCNC-mediated VAN removal.

[0189] FIG. 7 presents the effect of initial VAN concentration on AHCNC R.Interestingly, R ~ 10% at an initial VAN concentration of ~ 1 mg mL-1, despite the high qeof AHCNC. Nanoscale charged sorbents, such as AHCNC, do not undergo colloidal aggregation when the adsorbate concentration is stoichiometrically lower than the sorbent charged groups, i.e., the sorbent charge is not neutralized. Accordingly, the AHCNC-VAN aggregates remained colloidally stable in the solution when AHCNC were only partially saturated with VAN. R reached a maximum value (~ 48%) when the initial VAN concentration was - 4 mg mL-1, but decreased to 19% at 10 mg mL-1of VAN, because the active binding sites (i.e., dicarboxylate groups) of AHCNC were saturated with VAN. FIG. 8 shows VAN qeof AHCNC versus the equilibrium VAN concentration (Ce). The qeincreased as equilibrium VAN concentration increased to 4 mg mL-1, reaching a plateau of - 8316 ± 752 mg g-1.

[0190] Fl GS. 9-10 present the ^-potential and hydrodynamic equivalent size of AHCNC (1 mg mL-1) at varying initial VAN concentrations (pH = 6.5), respectively. The absolute value of AHCNC ^-potential decreased and reached - 0 by increasing the initial VAN concentration from 0 to 4 mg mL-1, confirming electrostatic interactions between AHCNC and VAN causing AHCNC charge neutralization. The hydrodynamic size of AHCNC did not significantly change (-100 nm) when the initial VAN concentrations increased from 0 to 2 mg mL-1; however, it reached > 2000 nm as a result of colloidal aggregation, and precipitates were observed at initial VAN concentrations > 4 mg mL-1.Atty. Ref. No. 0073605-000956

[0191] Fl G. 11 schematically shows the colloidal behavior of AHCNC sorbent when they are partially or fully saturated with VAN. Given that 1.11 mg mL’1of VAN neutralizes 0.25 mg mL’1of AHCNC, AHCNC were only partially saturated with VAN at initial VAN concentrations below 1.11 mg mb1. The partially saturated AHCNC remained colloidally stable in the solution without undergoing significant phase separation, and became fully saturated with VAN at the initial VAN concentrations > 1.11 mg mb1, inducing phase separation and precipitation. Accordingly, VAN is not completely removed by AHCNC at low VAN concentrations (Co < 1.11 mg mb1).Importantly, the physiological VAN concentration in the Gl tract is much lower than this value, limiting the in vivo translatability of AHCNC without using a substrate.

[0192] To overcome the colloidal stability limitation of AHCNC at low adsorbate-to-adsorbent ratios, it was coated on CHA to enable VAN separation at the low VAN concentrations. FIG. 12 presents the schematic of coating AHCNC on CHA via electrostatic interactions between AHCNC carboxylate groups and CHA quaternary ammonium groups, yielding a new bio-based sorbent, referred to as “AHCNC-CHA”. The binding energy between carboxylate groups and quaternary ammonium groups is ~ 71.2 kJ mol’1, resulting in AHCNC-CHA stability in the physiological conditions of Gl tract. FIG. 13 presents the optical microscopy images of CHA and AHCNC-CHA microscale particles. FIG. 1 summarize the concept of using AHCNC-CHA as an “antiantibiotic” adjuvant therapy versus not using it. If VAN remains in the intestine (FIG. 1), it may drive the VSEfm to resistant mutants, i.e., VREfm, which enter the bloodstream by moving across the intestinal lining and passing through the liver, causing infection. However, if anti-antibiotic is orally taken by IV VAN-administrated patients, it provides an orthogonal remedy to remove excess VAN from the Gl tract and to prevent the resistance pressure on the VSEfm, eliminating the emergence of VREfm.

[0193] Optimization of AHCNC-CHA anti-antibiotic composition: To maximize VAN removal capacity, AHCNC-CHA was fabricated at varying molar ratios of dicarboxylate groups of AHCNC to quaternary ammonium groups of CHA, ranging from 2:1 to 12:1, i.e., AHCNC concentrations varying from 3.3 mg mb1to 20 mg mL’1with aAtty. Ref. No. 0073605-000956constant CHA concentration of 20 mg mL-1. FIG. 14 presents the net carboxylate content of AHCNC-CHA and mass of adsorbed AHCNC, defined as the mass of adsorbed AHCNC to the mass of CHA, at varying COO' : quaternary ammonium (N+(CHS)3) molar ratios. The carboxylate content and mass of adsorbed AHCNC increased by increasing the molar ratio from 2 to 6 and reached a plateau (carboxylate content ~ 3 mmol g'1and mass of adsorbed AHCNC= 80%) at higher stoichiometric ratios, as a result of CHA saturation with AHCNC.

[0194] To examine the VAN removal performance of AHCNC-CHA compositions, a desired amount of each compound was incubated with VAN in varying media, as schematically shown in FIG. 15. FIG. 16 shows the VAN (1 mg mL'1) R of AHCNC-CHA prepared at varying COO' : (N+(CH3)3 stoichiometric ratios via changing varying AHCNC concentrations. R ~ 95% when the AHCNC-CHA stoichiometric ratio was above 6, resulting in the highest carboxylate group content, which electrostatically interacted with the amine groups of VAN. To assess the stability of AHCNC-CHA, FIG. 17 presents the R of AHCNC-CHA, prepared at varying COO' : (N+(CH3)s) stoichiometric ratios, after incubating AHCNC-CHA at varying pH ranging from 2 to 12 for 6 h (beyond the food residence time in the Gl tract). For convenience, we referred to the AHCNC-CHA formulated with varying stoichiometric ratios (SR) as AHCNC-CHA-SRn (n = 2, 4, 6, 8, 12). The Rfor all AHCNC-CHA decreased ~ 20% by decreasing pre-removal incubation pH from 6.5 to 1.5. This may be explained by two possibilities: (i) the “hairs” on AHCNC are partially hydrolyzed at extremely low pH; (ii) AHCNC is partially desorbed from the CHA as a result of carboxylate group partial protonation at extremely low pH. FIG. 18 shows the ratio of desorbed AHCNC mass from AHCNC-CHA with respect to initial AHCNC-CHA mass after incubating the AHCNC-CHA at varying pH for 6 h. As the desorbed AHCNC was < 5% at this pH range, the first possibility was more likely to be the main reason for the reduction in VAN removal.

[0195] Accordingly, AHCNC-CHA-SR6 bears the highest charge content, GR, R, and stability in acid media at the lowest possible AHCNC content. Therefore, it was selected as the optimal anti-antibiotic compound. For simplicity, we abbreviatedAtty. Ref. No. 0073605-000956AHCNC-CHA-SR6 as AHCNC-CHA using which the rest of this study was conducted. The VAN R of AHCNC-CHA after 6 h pre-removal incubation of AHCNC-CHA at pH = 1.5 was also studied and presented in FIG. 19. Although R decreased to 74% by decreasing pre-removal pH to 1.5 (FIG. 17), > 95% of VAN was removed by increasing AHCNC-CHA concentration (dose).

[0196] AHCNC-CHA was thoroughly characterized based on surface morphology, pore properties, and functional groups, as observed in FIGS. 20-22, via ATR-FTIR, SEM, and porosimetry, respectively. FIG. 20 shows the SEM images of CHA and AHCNC-CHA. The small width of nanocellulose (7 ± 2 nm, obtained from AFM images) renders the AHCNC visualization by SEM imaging non-trivial. CHA and AHCNC-CHA had similar particle size, topography, and surface features (wrinkles and rugged regions) within a micromere length scale. FIG. 21 presents the surface area, average pore diameter, and pore volume of CHA and AHCNC-CHA. The reduction in average diameter (d) and volume (i / ) of AHCNC-CHA (d = 30.9 nm, v = 0.00166 cm3g-1) pores compared with CHA (d = 57.3 nm, v = 0.00798 cm3g-1) may be a result of AHCNC adsorption to the surface and within the pores of CHA.

[0197] To confirm the AHCNC coating on CHA, FIG. 22 shows the ATR-FTIR spectra of AHCNC, CHA, and AHCNC-CHA. The peak at 3019 cm-1in CHA spectrum was attributed to the C-H stretching of quaternary ammonium groups. Shifting C-H stretching peak to 3030 cm-1in the AHCNC-CHA spectrum may be a result of the electrostatic binding of CHA quaternary ammonium groups to AHCNC carboxylate groups. The peak at 1602 cm-1in AHCHC spectrum was assigned to the stretching vibrations of carbonyl groups, pertaining to carboxylate (i.e., carboxyl group in the sodium form). In the AHCHC-CHA spectrum, this peak shifted to 1592 cm-1, likely because of the interactions of quaternary ammonium and carboxylate groups.

[0198] VAN removal at varying incubation times was studied to investigate the kinetics of AHCNC-CHA-mediated antibiotic capture. FIG. 23 shows the VAN R of AHCNC-CHA versus incubation time. The R reached ~ 40% almost immediately and increased to ~ 95% after 4 h of incubation. The time-dependent adsorption process mayAtty. Ref. No. 0073605-000956be associated with VAN molecular diffusion in the AHCNC-CHA pores, resulting from the porous structure of AHCNC-CHA. FIG. 24 presents the R at varying AHCNC-CHA doses, showing an increase from R ~ 7% to ~ 95% by increasing AHCNC-CHA dose from 0.8 to 20 mg mL’1, which confirms that the VAN removal process is dose dependent.

[0199] As the anti-antibiotic is an oral adjuvant, it must be able to pass through the stomach (pH ~ 1.5, 1 h < residence time < 4 h) before reaching the intestines.Therefore, the stability of AHCNC-CHA was assessed in the simulated gastric condition. FIG. 25 shows the desorption ratio of AHCNC from AHCNC-CHA after incubating in the SGF for varying periods. By increasing the incubation time from 1 h to 6 h, the desorption ratio increased from 4 ± 1% to 9 ± 2%, respectively. The AHCNC desorption ratio was less than 10% at the extreme acidic condition of SGF after 6 h. FIG. 26 presents the VAN R of SGF-incubated AHCNC-CHA. Although the R dropped by ~ 12% after 1 h and ~ 33% after 4 h of incubation in SGF because of the acid-mediated partial hydrolysis of poly anionic hairs, by increasing the dose of AHCNC-CHA to 60 mg mL’1, the R reached ~ 95%.

[0200] AHCNC-CHA-mediated VAN removal: To uncover the interactions between AHCNC-CHA and VAN, the effects of initial VAN concentrations, pH, and ionic strength on the R and qeof AHCNC-CHA were studied. FIG. 27 shows R and qeat varying initial VAN concentrations (1-30 mg mL’1). At initial VAN concentrations ranging from 1 to 8 mg mL’1, the R was ~ 95%. The molar stoichiometric ratio of AHCNC-CHA carboxylate groups to VAN amine groups was 10 : 0.125 at initial VAN concentration = 1 mg mL-1, which reached 10 : 1 at initial VAN concentration = 8 mg mL’1, enabling ~ 100% removal of VAN. When the initial VAN concentration increased beyond 8 mg mL’1, R decreased as a result of VAN-induced AHCNC-CHA carboxylate group saturation (neutralization). The qeincreased as the initial VAN concentration increased from 1 mg mL-1to 22 mg mL’1, reaching a plateau at ~ 850 mg g-1at higher initial VAN concentrations. The maximum qein this condition was ~ 38% lower than the theoretical value based on the charge stoichiometry, i.e., 3 mmol of AHCNC-CHA carboxylateAtty. Ref. No. 0073605-000956groups adsorbs 3 mmol (out of 5) of VAN amine groups, corresponding to 2239 mg g-1of VAN. The lower qecompared with the theoretical value may be because not all carboxylate binding sites on AHCNC-CHA electrostatically interacted with VAN as a result of steric hindrance.

[0201] FIG. 28 presents AHCNC-CHA-mediated VAN R at varying pH. By adjusting the pH to more acidic or alkaline conditions, R decreased. At pH < pKa,2 = 7.5, AHCNC-CHA carboxylate groups become protonated, decreasing the charge content and reducing the number density of binding sites. At pH > 7.5, the amine groups of VAN become deprotonated, reducing the positive charges, which in turn decreases the number of electrostatic binding sites. Accordingly, R decreased to ~ 65% at pH ~ 10, at which condition VAN and AHCNC-CHA bear net negative charges. The non-zero removal was possibly associated with the local positive charges on VAN, interacting with negatively charged AHCNC-CHA at pH ~ 10. These results suggested that the adsorption of VAN to AHCNC-CHA anti-antibiotic is governed by electrostatic attraction.

[0202] FIG. 29 shows the chemical structure of VAN, including the pKavalues of its major functional groups, as well as the net charge of VAN and AHCNC at varying pH based on their ionization states. The net charge of VAN is positive at pH ranging from 2-8, whereas its carboxylate groups are deprotonated, rendering the molecule anionic at pH > 8. FIGS. 30-31 present the ^-potential and hydrodynamic equivalent size of VAN and AHCNC at varying pH, respectively. The ^-potential of VAN changed from 20 ± 2 mV at pH = 2 to -33 ± 1 mV at pH = 12, justifying the reduction in R at alkaline pH (> 8) wherein the ^-potential of AHCNC was also negative. The hydrodynamic size of AHCNC and VAN changed from 89 ± 1 nm and 21 ± 6 nm at pH = 2, respectively, to 104 ± 2 nm and 49 ± 3 nm at pH =12.

[0203] Fl G. 32 shows the R and qeat varying sodium ion (Na+) concentrations. R did not significnatly change as the Na+concentration increased from 0 to 100 mM. This was explained by investigating the ^-potential and hydrodynamic size of VAN and AHCNC at varying Na+concentrations, as presented in FIGS. 33-34, respectively. The addition of Na+did not significantly change the ^-potential or hydrodynamic size of VANAtty. Ref. No. 0073605-000956and decreased the AHCNC ^-potential from -46 ± 1 to -31 ± 6 mV as a result of electrical double layer thickness reduction without compromising the AHCNC surface charge. Accordingly, the AHCNC-CHA anti-antibiotic remained charged and capable of binding VAN.

[0204] Fl G. 35 shows R and qeat varying Ca2+concentrations. Increasing the Ca2+concentration from 0 mM to 200 mM results in ~ 60% reduction in R as a result of AHCNC charge neutralization by the divalent cation (Ca2+: COO' = 1 mol : 2 mol).FIGS. 36-37 present the ^-potential and hydrodynamic equivalent size of VAN and AHCNC at varying Ca2+concentrations, respectively. The ^-potential (~ 14 ± 6 mV) and hydrodynamic size (~ 40 ± 7 nm) of VAN remain unchanged by increasing Ca2+concentration from 0 mM to 100 mM, which is because VAN and Ca2+are similarly charged. AHCNC ^-potential decreases from -46 ± 1 mV to -9 ± 1 mV, and the hydrodynamic equivalent size increases from 108 ± 1 nm to 6472 ± 791 nm, by increasing Ca2+concentration from 0 mM to 100 mM. These observations are attributed to Ca2+-mediated neutralization of AHCNC carboxylate groups and colloidal aggregation, respectively. Notably, ~ 42% of VAN is removed when the Ca2+concentration is ~ 200 mM, at which condition the AHCNC should theoretically be fully neutralized (30 mM of Ca2+stoichiometrically neutralizes 60 mM of carboxylate groups on 20 mg mL'1of AHCNC-CHA). The incomplete neutralization of AHCNC carboxylate groups may be attributed to the disordered dicarboxylate hairs closer to the crystalline region (inner hairs), which are likely less accessible or more structurally constrained compared to the outer hairs. To examine the effect of AHCNC-CHA dose on the antibiotic removal at the physiological concentration range of Ca2+(~ 2-6 mM), FIG. 38 presents VAN R at 5 mM of Ca2+and varying AHCNC-CHA doses. The initial reduction of R as a result of competitive Ca2+adsorption is compensated by increasing the AHCNC-CHA dose to 40 mg mL'1, yielding R ~ 95%.

[0205] In vitro AHCNC-CHA anti-antibiotic-mediated VAN removal in the SIF:SIF contains BA, MA, and PC, which may interact with VAN or AHCNC-CHA antiantibiotic. To emulate VAN removal from the intestines, we investigated the individualAtty. Ref. No. 0073605-000956and combined effects of SIF components on AHCNC-CHA-mediated VAN removal in vitro. FIG. 39 presents R at varying BA concentrations, ranging from 3 mM to 10 mM, i.e., the physiological BA concentration range in FaSSIF to FeSSIF. The R decreased by ~ 8% by increasing BA concentration form 0 mM to 10 mM as a result of the competition between BA and AHCNC-CHA anti-antibiotic to electrostatically bind VAN. The negatively charged sulfonate groups of BA may bind VAN, forming colloidally stable BA-VAN aggregates, thereby reducing R. Since the sulfonate groups are more electronegative than carboxylate, they have a higher affinity and binding strength to amine groups. Based on the charge stoichiometry, 10 mM of BA (containing 10 mM sulfonate groups) can electrostatically bind all VAN (1 mg mL-1= 1.379 mM protonated amine groups), which was in agreement with the experimental R (~ 92%).

[0206] FIG. 40 shows R at varying MA concentrations ranging from 5 mM to 100 mM (physiological MA concentration in SIF ~ 19 mM). The R decreased by ~ 23% at 20 mM MA because of the competition of carboxylate-bearing MA with AHCNC-CHA for VAN adsorption. FIG. 41 presents the R at 20 mM MA and varying AHCNC-CHA doses. The reduction in R was compensated by increasing the AHCNC-CHA dose to 60 mg mL’1, yielding R ~ 95%. When the MA concentration was above 30 mM, MA carboxylate concentration was higher than the AHCNC-CHA carboxylate content; however, the R still remained at ~ 60% at 100 mM MA, which may be associated with attractive van der Waals interactions between anionic AHCNC-CHA and nearly neutral MA-adsorbed VAN (^-potential = -8 ± 3 mV). This indicates that AHCNC-CHA could efficiently adsorb and remove VAN by inducing phase separation even when carboxylate-bearing competitors are in excess stoichiometric ratios.

[0207] FIG. 42 shows AHCNC-CHA-mediated VAN R at varying PC concentrations, ranging from 1 mM to 4 mM, i.e., the physiological concentration range from FaSSIF to FeSSIF. The R remained at ~ 95% for all PC concentrations. PC is zwitterionic, which is unable to effectively compete with AHCNC-CHA in VAN binding. These findings confirmed that VAN adsorption to AHCNC-CHA is feasible in the intestines.Atty. Ref. No. 0073605-000956

[0208] Fl G. 43 presents R at varying initial VAN concentrations in the FaSSIF and FeSSIF. R ~ 95% when the initial VAN concentration was < 2 mg mb1in both conditions; however, R in the FeSSIF was always slightly lower than that in the FaSSIF at initial VAN concentrations of > 2 mg mb1, which was likely a result of the higher BA and MA concentrations in the FeSSIF. FIG. 44 shows qeat varying initial VAN concentrations in the FaSSIF and FeSSIF. The qein FaSSIF (~ 380 mg g-1) and in FeSSIF (~ 340 mg g-1) were ~ 50% lower than that in Milli-Q water (~ 860 mg g-1). The lower qein FaSSIF and FeSSIF was possibly caused by competitive adsorption induced by BA and MA, particularly MA, decreasing R up to ~ 23% at 19 mM MA (FaSSIF) and ~ 35% in 55 mM MA (FeSSIF) (FIG. 40).

[0209] In vitro assessment of VAN activity after AHCNC-CHA-mediated VAN removal: To evaluate the bioactivity of VAN after incubation with AHCNC-CHA, its antibacterial efficacy was tested against VSE / m in the broth microdilution assay at initial VAN concentrations ranging from 0.125 to 8 pg mL-1. FIG. 45 presents the ODeoo of bacteria following growth in supernatants collected from a VAN solution treated with AHCNC, CHA, or AHCNC-CHA anti-antibiotic independently. The ODeoo decreased up to 3 orders of magnitude by increasing the initial VAN concentration from 0.5 to 8 pg mb1when AHCNC or CHA was used, similar to untreated VAN solution, as these compounds were not able to remove VAN from the supernatants; however, the incubation of AHCNC-CHA anti-antibiotic with VAN resulted in no antibiotic activity against VSEfm. It was observed that AHCNC alone did not induce sufficient colloidal aggregation and phase separation, leaving a large fraction of VAN in the supernatant by which the bacteria were killed. AHCNC-CHA completely captured VAN via phase separation; thus, no antibiotic activity was observed. The AHCNC-CHA removal efficacy at physiologically relevant low VAN concentrations was proved using HPLC analysis. FIG. 46 shows Rat initial VAN concentrations below 100 pg mL-1. VAN was almost completely removed at the low antibiotic concentrations.

[0210] Fl G. 47 presents the ODeoo of bacteria following growth in the supernatants obtained from VAN solutions incubated with AHCNC-CHA anti-antibiotic atAtty. Ref. No. 0073605-000956varying periods. After 1 h of VAN contact with AHCNC-CHA, no antibiotic activity is detected against VSEfrn, implying that the available AHCNC on the surface of CHA swiftly remove VAN. FIG. 48 shows the ODeoo of bacteria following growth in the supernatants treated with varying AHCNC-CHA doses for 4 h. The available antibiotic in the supernatant decreases by increasing the AHCNC-CHA dose, resulting in less antibiotic activity against VSEfrn and an increase in the bacterial density. To evaluate the VAN removal efficacy of AHCNC-CHA exposed to strong acid, mimicking the gastric pH, at physiological VAN concentrations, FIG. 49 presents the ODeoo of bacteria following growth in supernatants collected from a VAN solution incubated with acid (5 M HCI)-treated AHCNC-CHA for 4 h, implying no antibiotic activity against VSEfrn. The results suggest that although the acid-incubated AHCNC-CHA anti-antibiotic undergoes a reduction in VAN (1 mg mL-1) R (shown in FIG. 17), it is able to completely capture VAN in the physiological VAN concentrations.

[0211] In vivo AHCNC-CHA anti-antibiotic efficacy assessment: To assess the in vivo efficacy of AHCNC-CHA anti-antibiotic in reducing the pressure on resistance selection, a murine model of intestinal E. faecium colonization was used. FIGS. 50-51 show the fecal densities of total E. faecium in mice, treated with saline or either 4 or 10 mg of AHCNC-CHA anti-antibiotic. Prior to bacterial inoculation, no mice had detectable E. faecium in their feces. Following inoculation with a 1 : 20 ratio of VREfm : VSEfm, treatment of mice with either 4 or 10 mg of AHCNC-CHA anti-antibiotic did not significantly change the total E. faecium in the feces or the body mass of the mice (FIGS. 52-53) relative to the saline-treated controls. FIGS. 54-55 present the fecal densities of VREfm in mice treated with saline or either 4 or 10 mg of anti-antibiotic. In contrast, following subcutaneous VAN injection, mean fecal VREfrn density decreased by ~ 46% and ~ 63% for the mice treated with 4 mg or 10 mg AHCNC-CHA antiantibiotic, respectively, relative to the saline-treated controls. These differences were statistically significant in mice treated with 10 mg of AHCNC-CHA. FIGS. 56-57 show the fecal densities of VSEfrn in mice treated with saline or either 4 or 10 mg of antiantibiotic. The decrease in VREfrn corresponded with an anti-antibiotic dose-dependentAtty. Ref. No. 0073605-000956increase in mean VSE / m fecal shedding in mice treated with either 4 mg (by ~ 90%) or 10 mg (by ~ 194%) of AHCNC-CHA anti-antibiotic. This again was most pronounced in mice treated with 10 mg of AHCNC-CHA.

[0212] To further explore these relative changes, the proportion of VREfm was calculated for each fecal sample. FIGS. 58-59 present the proportion of VREfm as a function of post-inoculation time and the cumulative portion of VRE / im in mice treated with saline or either 4 or 10 mg of AHCNC-CHA anti-antibiotic, respectively. Compared with the saline-treated controls, the mice treated with 10 mg of AHCNC-CHA antiantibiotic twice daily underwent a significant decrease in the cumulative proportion of fecal VREfm relative to VSEfrn (from ~ 77% for saline treated mice to ~ 29% for mice treated with 10 mg of AHCNC-CHA anti-antibiotic). Taken together, these data suggested that the AHCNC-CHA effectively sequesters VAN in the murine Gl tract, reducing the pressure on resistance selection.

[0213] Based on our conducted evaluation work discussed herein, it has been shown that embodiments of our process can remove off-target VAN from the Gl tract after intravenous VAN administration, and can effectively prevent the evolution of transmissible VAN-resistant pathogens.

[0214] 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.Atty. Ref. No. 0073605-000956

[0215] 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.

[0216] 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

Atty. Ref. No. 0073605-000956WHAT IS CLAIMED IS:

1. An antibiotic removal or deactivation agent, comprisinghairy cellulose nanocrystals;a particulate resin; andoptionally, a functionalized biopolymer,wherein the hairy cellulose nanocrystals are immobilized on the particulate resin.

2. The antibiotic removal or deactivation agent of claim 1 , wherein the particulate resin is comprised of cholestyramine, 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.

3. The antibiotic removal or deactivation agent of claim 1 , wherein the particulate resin is cholestyramine.

4. The antibiotic removal or deactivation agent of claim 1 , wherein the hairy cellulose nanocrystals comprise cellulose bodies bearing cellulose chains protruding therefrom, wherein the cellulose chains are functionalized with anionic groups.

5. The antibiotic removal or deactivation agent of claim 4, wherein the anionic groups comprise carboxylate groups.Atty. Ref. No. 0073605-0009566. The antibiotic removal or deactivation agent of claim 1 , wherein the hairy cellulose nanocrystals comprise cellulose bodies bearing cellulose chains protruding therefrom, wherein the cellulose chains are functionalized with cationic groups.

7. The antibiotic removal or deactivation agent of claim 1 , wherein the hairy cellulose nanocrystals comprise cellulose bodies bearing cellulose chains protruding therefrom, wherein the cellulose chains are functionalized with amphiphilic groups.

8. The antibiotic removal or deactivation agent of claim 1 , wherein the hairy cellulose nanocrystals comprise cellulose bodies bearing cellulose chains protruding therefrom, wherein the cellulose chains are functionalized with zwitterionic groups.

9. The antibiotic removal or deactivation agent of claim 1 , wherein the hairy cellulose nanocrystals comprise cellulose bodies bearing cellulose chains protruding therefrom, wherein the cellulose chains are functionalized with a combination of electrically neutral anionic, cationic, zwitterionic, and / or amphiphilic groups.

10. The antibiotic removal or deactivation agent of claim 1 , wherein the functionalized biopolymer comprises polysaccharide, protein, polynucleotides, and / or polypeptides, wherein the biopolymer is functionalized with a combination of electrically neutral anionic, cationic, zwitterionic, and / or amphiphilic groups.

11. The antibiotic removal or deactivation agent of claim 1 , wherein the agent is configured to be administered to a gastrointestinal (Gl) tract of a patient, such that when the Gl tract includes one or more antibiotic compounds, the agent is configured to effectively remove the one or more antibiotic compounds.

12. The antibiotic removal or deactivation agent of claim 11 , wherein the one or more antibiotic compounds include B-lactams, aminoglycosides, chloramphenicol,Atty. Ref. No. 0073605-000956glycopeptides, quinolones, oxazolidinones, sulfonamides, tetracyclines, macrolides, ansamycins, streptogramins, lipopeptides, or combinations thereof.

13. The antibiotic removal or deactivation agent of claim 11 , wherein the one or more antibiotic compounds include Vancomycin (VAN).

14. An antibiotic removal or deactivation agent, comprisinga functionalized biopolymer, wherein the functionalized biopolymer comprises polysaccharide, protein, polynucleotides, and / or polypeptides, wherein the polymer is functionalized with a combination of electrically neutral anionic, cationic, zwitterionic, and / or amphiphilic groups.

15. The antibiotic removal or deactivation agent of claim 14, wherein the agent is configured to be administered to a gastrointestinal (Gl) tract of a patient, such that when the Gl tract includes one or more antibiotic compounds, the agent is configured to effectively remove the one or more antibiotic compounds.

16. The antibiotic removal or deactivation agent of claim 15, wherein the one or more antibiotic compounds include B-lactams, aminoglycosides, chloramphenicol, glycopeptides, quinolones, oxazolidinones, sulfonamides, tetracyclines, macrolides, ansamycins, streptogramins, lipopeptides, or combinations thereof.

17. The antibiotic removal or deactivation agent of claim 15, wherein the one or more antibiotic compounds include Vancomycin (VAN).

18. A process for removing one or more antibiotic compounds from a gastrointestinal (Gl) tract of a patient, comprising:Atty. Ref. No. 0073605-000956administering an antibiotic removal or deactivation agent to the patient for digestion so that the antibiotic removal agent or the antibiotic deactivation agent is present in the Gl tract of the patient,wherein the antibiotic removal or deactivation agent effectively removes the one or more antibiotic compounds present in the Gl tract, andwherein the antibiotic removal or deactivation agent comprises hairy cellulose nanocrystals immobilized on a particulate resin.

19. The process of claim 18, wherein the particulate resin is comprised of cholestyramine, 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.

20. The process of claim 18, wherein the particulate resin comprises cholestyramine.

21. The process of claim 18, wherein the one or more antibiotic compounds include B-lactams, aminoglycosides, chloramphenicol, glycopeptides, quinolones, oxazolidinones, sulfonamides, tetracyclines, macrolides, ansamycins, streptogramins, lipopeptides, or combinations thereof.

22. The process of claim 18, wherein the one or more antibiotic compounds include Vancomycin (VAN).

23. The process of claim 18, wherein the antibiotic removal or deactivation agent effectively removing the one or more antibiotic compounds present in the Gl tract includes the antibiotic removal or deactivation agent interacting with the one or moreAtty. Ref. No. 0073605-000956antibiotic compounds present in the Gl tract such that one or more antibacterial compounds present in the Gl tract are non-bioreactive with bacteria within the Gl tract.

24. The process of claim 18, further comprising:treating the patient with the one or more antibiotic compounds to treat an infectious disease in the patient.

25. The process of claim 24, wherein the infectious disease is a bacterial infection that is not located in the Gl tract.

26. The process of claim 24, wherein the infectious disease is a bacterial infection that is located in the Gl tract.

27. The process of claim 18, further comprising:treating the patient with the one or more antibiotic compounds to treat a non-infectious disease in the patient.

28. The process of claim 27, wherein the non-infectious disease is a bacterial infection that is not located in the Gl tract.

29. The process of claim 27, wherein the non-infectious disease is a bacterial infection that is located in the Gl tract.

30. The process of claim 18, wherein the administering of the antibiotic removal or deactivation agent to the patient for the patient to digest so that the antibiotic removal or deactivation agent is present in the Gl tract of the patient comprises:feeding the patient the antibiotic removal or deactivation agent such that the antibiotic removal or deactivation agent is orally consumed by the patient.Atty. Ref. No. 0073605-00095631. The process of claim 18, wherein the administering of the antibiotic removal or deactivation agent to the patient for the patient to digest so that the antibiotic removal or deactivation agent is present in the Gl tract of the patient comprises:feeding the patient the antibiotic removal or deactivation agent periodically while the patient undergoes a treatment for an infection that includes multiple injections of the one or more antibiotic compounds into the patient at different spaced apart intervals.

32. The process of claim 18, wherein the administering is performed periodically different times a day for a number of days.

33. The process of claim 18, wherein the antibiotic removal or deactivation agent effectively removes the one or more antibiotic compounds present in the Gl tract such that bacteria within the Gl tract is unaffected by the antibiotic compounds.

34. The process of claim 18, wherein the antibiotic removal or deactivation agent effectively removes the one or more antibiotic compounds present in the Gl tract to reduce, hinder, or avoid the growth or development of drug resistant bacteria and / or the protection of drug sensitive bacteria.

35. The process of claim 18, wherein the antibiotic removal or deactivation agent effectively removes the one or more antibiotic compounds present in the Gl tract to block or avoid antibiotic-resistant bacterial growth within the body.

36. The process of claim 18, wherein the antibiotic removal or deactivation agent effectively removes the one or more antibiotic compounds present in the Gl tract to help protect or promote antibiotic-sensitive bacterial growth within the body.Atty. Ref. No. 0073605-00095637. The process of claim 18, wherein the antibiotic removal or deactivation agent effectively removes the one or more antibiotic compounds present in the Gl tract to help protect the microbiome within the body.

38. A process for removing one or more antibiotic compounds from a gastrointestinal (Gl) tract of a patient, comprising:administering an antibiotic removal or deactivation agent to the patient for digestion so that the antibiotic removal agent or the antibiotic deactivation agent is present in the Gl tract of the patient,wherein the antibiotic removal or deactivation agent effectively removes the one or more antibiotic compounds present in the Gl tract, andwherein the antibiotic removal or deactivation agent comprises a functionalized biopolymer, wherein the functionalized biopolymer comprises polysaccharide, protein, polynucleotides, and / or polypeptides, wherein the polymer is functionalized with a combination of electrically neutral anionic, cationic, zwitterionic, and / or amphiphilic groups.

39. The process of claim 38, wherein the particulate resin is comprised of cholestyramine, 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.

40. The process of claim 38, wherein the particulate resin comprises cholestyramine.

41. The process of claim 38, wherein the one or more antibiotic compounds include B-lactams, aminoglycosides, chloramphenicol, glycopeptides, quinolones,Atty. Ref. No. 0073605-000956oxazolidinones, sulfonamides, tetracyclines, macrolides, ansamycins, streptogramins, lipopeptides, or combinations thereof.

42. The process of claim 38, wherein the one or more antibiotic compounds include Vancomycin (VAN).

43. The process of claim 38, wherein the antibiotic removal or deactivation agent effectively removing the one or more antibiotic compounds present in the Gl tract includes the antibiotic removal or deactivation agent interacting with the one or more antibiotic compounds present in the Gl tract such that one or more antibacterial compounds present in the Gl tract are non-bioreactive with bacteria within the Gl tract.

44. The process of claim 38, further comprising:treating the patient with the one or more antibiotic compounds to treat an infectious disease in the patient.

45. The process of claim 44, wherein the infectious disease is a bacterial infection that is not located in the Gl tract.

46. The process of claim 44, wherein the infectious disease is a bacterial infection that is located in the Gl tract.

47. The process of claim 38, further comprising:treating the patient with the one or more antibiotic compounds to treat an non-infectious disease in the patient.

48. The process of claim 47, wherein the infectious disease is a bacterial infection that is not located in the Gl tract.Atty. Ref. No. 0073605-00095649. The process of claim 47, wherein the infectious disease is a bacterial infection that is located in the Gl tract.

50. The process of claim 38, wherein the administering of the antibiotic removal or deactivation agent to the patient for the patient to digest so that the antibiotic removal or deactivation agent is present in the Gl tract of the patient comprises:feeding the patient the antibiotic removal or deactivation agent such that the antibiotic removal or deactivation agent is orally consumed by the patient.

51. The process of claim 38, wherein the administering of the antibiotic removal or deactivation agent to the patient for the patient to digest so that the antibiotic removal or deactivation agent is present in the Gl tract of the patient comprises:feeding the patient the antibiotic removal or deactivation agent periodically while the patient undergoes a treatment for an infection that includes multiple injections of the one or more antibiotic compounds into the patient at different spaced apart intervals.

52. The process of claim 38, wherein the administering is performed periodically different times a day for a number of days.

53. The process of claim 38, wherein the antibiotic removal or deactivation agent effectively removes the one or more antibiotic compounds present in the Gl tract such that bacteria within the Gl tract is unaffected by the antibiotic compounds.

54. The process of claim 38, wherein the antibiotic removal or deactivation agent effectively removes the one or more antibiotic compounds present in the Gl tract to reduce, hinder, or avoid the growth or development of drug resistant bacteria and / or the protection of drug sensitive bacteria.Atty. Ref. No. 0073605-00095655. The process of claim 38, wherein the antibiotic removal or deactivation agent effectively removes the one or more antibiotic compounds present in the Gl tract to block or avoid antibiotic-resistant bacterial growth within the body.

56. The process of claim 38, wherein the antibiotic removal or deactivation agent effectively removes the one or more antibiotic compounds present in the Gl tract to help protect or promote antibiotic-sensitive bacterial growth within the body.

57. The process of claim 38, wherein the antibiotic removal or deactivation agent effectively removes the one or more antibiotic compounds present in the Gl tract to help protect the microbiome within the body.

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