COMPOUNDS AND PHARMACEUTICAL COMPOSITIONS FOR PREVENTION AND/OR TREATMENT OF DYSBIOSIS AND ANTIBACTERIAL ANTIDOTES FOR MICROBIOME-PROTECTION

Abstract

The present invention relates to the field of therapeutics and, more in particular, to pharmaceutical compositions for the prevention and/or treatment of bacterial infections and antibacterial-induced dysfunctions. The invention is based on the identification of compounds that prevent antibacterial effects of antibiotics on microbiome-bacterial species, while retaining antibacterial effects of antibiotics on pathogenic bacteria. The invention also pertains to pharmaceutical compositions comprising antibiotics and antibiotic antidotes for the protection of commensal microbiome-bacteria. Thus, this invention relates to compounds and pharmaceutical combinations useful to prevent antibiotic-induced adverse effects on the gut microbiome. Furthermore provided are methods for preventing an adverse effect on a gut microbiome using the compounds and/or pharmaceutical compositions of this invention.

Claims

1. A compound, or a pharmaceutically acceptable salt thereof, for use in treating and/or preventing dysbiosis, and/or for use in antagonizing an antibacterial effect of at least one second compound in at least one first bacterium, wherein said compound, or the pharmaceutically acceptable salt thereof, is capable of antagonizing an antibacterial effect of at least one second compound in at least one first bacterium, and wherein said compound, or the pharmaceutically acceptable salt thereof, is not capable of antagonizing an antibacterial effect of said at least one second compound in at least one second bacterium.

2. (canceled)

3. A pharmaceutical composition, comprising: (i) at least one first compound, or a pharmaceutically acceptable salt thereof, wherein said at least one first compound is capable of antagonizing an antibacterial effect of at least one second compound in a first bacterium, and wherein said at least one first compound is not capable of antagonizing an antibacterial effect of said at least one second compound in a second bacterium; (ii) at least one second compound, or a pharmaceutically acceptable salt thereof, and (iii) optionally, a pharmaceutically acceptable additive, carrier, diluent, solvent, filter, lubricant, excipient, binder, and/or stabilizer.

4. The pharmaceutical composition according to claim 3, wherein said at least one first compound (i) is selected from an anticoagulant drug, a non-steroidal anti-inflammatory drug, an Uricosuric, a nonsteroidal estrogen compound, an antagonist of the Cholecystokinin CCKA receptor, a salicylic acid derivative, a cholecystographic agent, an angiotensin II receptor blocker, a thyroid hormone analogue, an antibacterial agent, antifungal agent and/or antibacterial agent, a reverse transcriptase inhibitor, a Ca-channel inhibitor, a human-targeted drug, and a food additive, or a pharmaceutically acceptable salt thereof.

5. The pharmaceutical composition according to claim 3, wherein said at least one second compound is an antibiotic.

6. The pharmaceutical composition according to claim 3, wherein said at least one first compound (i) is Dicumarol, Tolfenamic acid, Benzbromarone, Diethylstilbestrol, Famprofazone, Hexestrol, or Lorglumide, or a pharmaceutically acceptable salt thereof, and said at least one second compound or a pharmaceutically acceptable salt thereof.

7. The pharmaceutical composition according to claim 3, wherein said at least one first compound (i) is Diflunisal, Tolfenamic acid, Iopanoic acid, Tiratricol, Celecoxib, Losartan, Triclosan, Clonixin, Diethylstilbestrol, Meclofenamic acid, Diclazuril, Efavirenz, or a pharmaceutically acceptable salt thereof, and said at least one second compound (ii) is an antibiotic.

8. The pharmaceutical composition according to claim 3, wherein said at least one first bacterium is a member of a genus selected from Bacteroides, Bacteroidales, Eubacterium, Faecalibacterium, Odoribacter, Prevotella, Parabacteroides, Ruminococcus, Roseburia, Bifidobacterium, Coprococcus, Dorea, Blautia, Clostridium, Escherichia, Streptococcus, Collinsella, Bilophila, Akkermansia, Veillonella, Haemophilus, Desulfovibrio, Fusobacterium, Lactobacillus, Alistipes, and Butyrivibrio.

9. The pharmaceutical composition according to claim 3, wherein said at least one second bacterium is a member of a genus selected from Enterococcus, Staphylococcus, Enterobacter, Streptococcus, Pseudomonas, Escherichia, Salmonella, Helicobacter, Neisseria, Campylobacter, Chlamydia, Clostridia, Citrobacter, Vibrio, Treponema, Mycobacterium, Klebsiella, Actinomyces, Bacteroides, Bordetella, Borrelia, Brucella, Corynebacteria, Diplococcus, Fusobacterium, Leptospira, Listeria, Pasteurella, Proteus, Rickettsia, Shigella, Yersinia, Parabacteroides, Odoribacter, Faecalibacteria, Collinsella, Eggerthella, Lactonifactor, Pediococcus, Leuconostoc, Lactococcus, Roseburia, Coliform, Bacillus, Franscicella, Acinetobacter, Legionella, Actinobacillus, Coxiella, Kingella kingae, and Haemophilus.

10. The pharmaceutical composition according to claim 3, wherein said at least one second compound is for use in the prevention and/or treatment of a bacterial infection is selected from an infection of the gastrointestinal tract, an infection of the urogenital tract, an infection of the upper and lower respiratory tract, rhinitis, tonsillitis, pharyngitis, bronchitis, pneumonia, an infection of the inner organs, nephritis, hepatitis, peritonitis, endocarditis, meningitis, osteomyelitis, an infection of the eyes, an infection of the ears, a cutaneous infection, a subcutaneous infection, an infection after burn, diarrhea, colitis, pseudomembranous colitis, a skin disorder, toxic shock syndrome, bacteremia, sepsis, pelvic inflammatory disease, an infection of the central nervous system, wound infection, intra-abdominal infection, intravascular infection, bone infection, joint infection, acute bacterial otitis media, pyelonephritis, deep-seated abscess, and tuberculosis.

11. The pharmaceutical composition according to claim 3, wherein said pharmaceutical composition is in the form of a tablet, coated tablet, effervescent tablet, capsule, powder, granulate, sugar-coated tablet, lozenge, pill, ampoule, drop, suppository, emulsion, ointment, gel, tincture, paste, cream, moist compress, gargling solution, plant juice, nasal agent, inhalation mixture, aerosol, mouthwash, mouth spray, nose spray, or room spray.

12-14. (canceled)

15. A method for preventing an adverse effect on a gut microbiome using a compound according to claim 1, or a pharmaceutical composition comprising: (i) at least one first compound, or a pharmaceutically acceptable salt thereof, wherein said at least one first compound is capable of antagonizing an antibacterial effect of at least one second compound in a first bacterium, and wherein said at least one first compound is not capable of antagonizing an antibacterial effect of said at least one second compound in a second bacterium; (ii) at least one second compound, or a pharmaceutically acceptable salt thereof, and (iii) optionally, a pharmaceutically acceptable additive, carrier, diluent, solvent, filter, lubricant, excipient, binder, and/or stabilizer, the method comprising administering to a subject an effective amount of said compound and/or said pharmaceutical composition, thereby treating the disease in the subject.

16. The pharmaceutical composition according to claim 5, wherein said antibiotic is selected from a macrolide, a penicillin, a cephalosporin, a quinolone, a fluoroquinolone, a sulphonamide, a tetracycline, a monobactam, a carbapnem, an aminoglycoside, a rifamycin, a beta-lactam, an ansamycin, an oxazolidinone, a strepotgramin, a glycopeptide, a polypeptide, and an arsphenamine, or a pharmaceutically acceptable salt thereof.

17. The pharmaceutical composition according to claim 5, wherein said antibiotic is selected from erythromycin, azithromycin, clarithromycin, dirithromycin, clindamycin, doxycycline, minocycline, tigecyline, trimethoprim, pyocyanin, vancomycin, streptomycin, dihydrostreptomycin, amikacin, apramycin, arbekacin, astromicin, bekanamycin, dibekacin, framycetin, gentamicin, hygromycin, isepamicin, kanamycin, neomycin, netilmicin, paromomycin, rhodostreptomycin, ribostamycin, sisomycin, spectinomycin, tobramycin, verdamicin, polymixin, glycylcycline, carbapenem, carbacephem, chloramphenicol, clindamycin, lincomycin, daptomycin, novobiocin, clindamycin, ethambutol, fosfomycine, fusidic acid, furazolidone, isoniazid, linezolide, metronidazole, mupirocin, nitrofurantoin, platensimycine, pyrazinamide, quinupristine, benzalkonium, dalfopristine, rifampine, rifampicin, rifabutin, rifaximin, tinidazole, viomycin, and capreomycin, or a pharmaceutically acceptable salt thereof.

18. The pharmaceutical composition according to claim 6, wherein said macrolide is azithromycin, clarithromycin, dirithromycin, clindamycin, or a pharmaceutically acceptable salt thereof.

19. The pharmaceutical composition according to claim 7, wherein said antibiotic is doxycycline, minocycline, tigecyline, lymecyclin, or a pharmaceutically acceptable salt thereof.

20. The method according to claim 15, wherein said subject is a human.

21. The method according to claim 15, wherein said compound or said pharmaceutical composition is administered to a subject, thereby modifying the growth of bacterial cells in said subject, wherein said modifying results in the prevention and/or treatment of a disease in said subject, and/or in the prevention of a modification of the microbiome of said subject.

22. The method according to claim 20, wherein the disease is dysbiosis.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0171] The figures show:

[0172] FIG. 1 shows macrolides and tetracyclines kill human gut commensal species. a. The survival of 12 abundant gut microbe species was measured after a 5-hour treatment with a 5-fold MIC of erythromycin (ERY), azithromycin (AZI) or doxycycline (DOX). The survival was assessed by counting CFUs/ml before and after antibiotic treatment. The number of CFUs/ml before treatment was set as 100%. The detection limit for each experiment (gray bar) and the bactericidal threshold (shaded area) are indicated. Species are plotted according to phylogeny (IQTree, Methods) and in bold are noted the species that are used in later panels. The graph shows the mean+SD of 3 independent experiments. b. Time-kill curves of B. vulgatus, R. intestinalis and F. nucleatum after antibiotic treatments. Survival was assessed by CFU counting over a 5 hour-treatment of ERY, AZI or DOX. This graph shows the mean±SD of 3 independent experiments. Nd: non-detectable. Time-kill curves for the other tested gut microbes can be found in FIG. 2. C. Erythromycin induces lysis of B. vulgatus and B. uniformis. B. vulgatus and B. uniformis were grown for 3 hours before addition (yellow) or not (black) of 15 μg/ml ERY treatment (5-fold MIC; yellow) as indicated by the arrow. Growth curves were acquired for 20 hours. This graph shows the mean±SD (dotted line) of 3 independent experiments. d. Live/dead staining of macrolide or tetracycline-treated B. vulgatus. The left panel shows an overlay of phase contrast and fluorescence microscopy images of propidium iodide (PI)-stained B. vulgatus before and 5 hrs after ERY, AZI or DOX treatment. Cultures were concentrated before imaging; the scale bar is 10 μm. The right panel shows the corresponding quantification of live/dead-stained cells by flow cytometry with Syto9 on the x-axis (live cells) and PI on the y-axis (dead cells). Both the total number of measured events (n) and the percentage of cells found in each quadrant are indicated. e. Erythromycin induces blebbing, cytoplasmic shrinkage and lysis in B. vulgatus and B. uniformis. Phase contrast movies of B. vulgatus and B. uniformis were acquired after ERY treatment (5-fold MIC). Here shown are 3 frames of 3 images per strain (time indicated in the upper left corner; t=0 when drug added). White arrows indicate blebs, cytoplasmic shrinkage and bacterial lysis; the scale bar is 5 μm.

[0173] FIG. 2 shows time-kill curves of 12 abundant gut microbes after treatment with macrolides and tetracyclines. Survival of 12 abundant gut microbes was assessed by CFU counting over a 5 hour-treatment of either ERY, AZI or DOX. This graph shows the mean±SD of 3 independent experiments.

[0174] FIG. 3 shows antidotes for selective protection of prevalent and abundant gut commensal species from macrolides and tetracyclines. a. Schematic illustration of the screen concept: searching for antidote compounds that antagonize the antibacterial effect of erythromycin or doxycycline on commensal but not on pathogenic bacteria. b. Z-scores on bacterial growth (based on areas under the curve (AUCs)) for combinatorial drug exposure with antibiotic (ERY or DOX) and FDA-approved drug. Compounds that successfully rescued B. vulgatus and/or B. uniformis growth in the presence of the antibiotic (z-score >3) are indicated in gray. The strongest hits (circles) were validated further in concentration-dependent assays (FIG. 5 a). For each antibiotic and each strain, ˜1200 drugs were tested in two replicates. Boxplots are plotted as in FIG. 1 d. c. For 9 of the validated antagonists, 8×8 checkerboard assays were performed to determine concentration ranges of the antagonistic interaction. Heat maps depict bacterial growth based on normalized median of AUCs of 4 replicates. Antagonistic interactions are framed in red, and pairs tested further in other commensal species in bold. d. Checkerboard assays confirm the ability of tolfenamic acid to protect further gut commensals from growth inhibition by erythromycin. Heat map as in c, but for 2 replicates. Antagonistic interactions are framed in red. e. Checkerboard of tolfenamic acid with erythromycin reveal neutral interactions in S. aureus and E. faecium (aerobic conditions). Heat maps as in c. f. Tolfenamic acid concentration-dependent rescue of commensal growth at clinical relevant erythromycin concentrations based on AUCs (anaerobic conditions). Erythromycin still retains its activity against pertinent pathogens such as S. aureus and S. pneumoniae (aerobic conditions). 0.625 μM correspond to ˜0.5 μg/ml erythromycin, which is in the range of the MIC breakpoints for Staphylococcus (1 μg/ml), S. pneumoniae (0.25 μg/ml) and Streptococcus groups A, B, C & G (0.25 μg/ml). Error bars depict standard deviation.

[0175] FIG. 4 shows a schematic overview of the screen for microbiome-protective antibiotic antagonisms. Workflow with decision process which antagonist to move on to next evaluation step.

[0176] FIG. 5 shows validation of potential microbiome-protective antagonists. a. Validation of the strongest antagonists in independent experiments. Erythromycin and doxycycline concentrations were kept constant ([ERY]=0.625 μM, [DOX]=0.039/0.078 μM) and concentration ranges were tested for antagonist. Asterisks indicate that at least 25% of the bacterial growth (compared to no drug controls) could be rescued by the antagonist at a given concentration. Heat map depicts median AUCs across triplicates. b. Percentage of surviving B. vulgatus cells were determined after 5 h incubation with either erythromycin (3.25 μM) or doxycycline (0.4 μM) alone or in presence of the antagonist dicumarol (20 μM), tolfenamic acid (40 μM) or diflunisal (80 μM). Data is based on 3 replicates. Boxplots are plotted as in FIG. 1 d.

[0177] FIG. 6 shows the effect of antidotes on further gut commensals. 8×8 checkerboard assays to investigate if antidote is also protective for additional gut commensals for the following combinations: erythromycin and dicumarol (a), doxycycline and diflunisal (b) and doxycycline and tolfenamic acid (c). Heat map depicts bacterial growth based on median AUCs from two independent replicates. Red contours indicate antagonistic drug interactions.

[0178] FIG. 7 shows the effect of the antidote dicumarol on pathogens, relatively to commensal species. a. Checkerboard assays for the drug combinations erythromycin-tolfenamic acid and erythromycin-dicumarol on the pathogens S. aureus (two different strains) and E. faecium. Heat map depict median normalized AUCs of checkerboard assays (at least three independent replicates). b. Dicumarol rescues commensal growth (based on median AUCs, N=2) at clinical relevant erythromycin concentrations in a concentration-dependent manner. Erythromycin still retains its activity against pertinent pathogens such as S. aureus and S. pneumoniae (based on median AUCs, N=3). Error bars depict standard deviation.

[0179] FIG. 8 shows the effect of the antidote dicumarol, benzbromarone or tolfenamic acid on azithromycin treatment of species of the order bacteroidales (Example 4). The antidotes dicumarol (first column), benzbromarone (second column) and tolfenamic acid (third column) were tested for their efficacy in antagonizing the macrolide antibiotic azithromycin across seven prevalent and abundant gut Bacteroidales (B. vulgatus, B. fragilis NT, B. thetaiotaomicron, P. copri, B. caccae, B. ovatus, P. distasonis). Heat maps depict bacterial growth based on normalized median of the area under the growth curve (AUCs) of 3 replicates. All drug concentrations are in μM.

[0180] FIG. 9 shows that synthetic gut commensal communities can be protected from macrolide antibiotics by the antidotes dicumarol, benzbromarone or tolfenamic acid (Example 5). Heat maps depict area under the growth curve (AUC) of a synthetic community consisting of seven human gut commensals. Shown is the median AUC of three individual replicates. All drug concentrations are in μM.

[0181] FIG. 10 shows that human intestinal pathogens of the genus enterococcus are not rescued from macrolide antibiotics by the antidotes dicumarol, benzbromarone or tolfenamic acid (Example 6). Heat maps show median area under the growth curve (AUC) of three biologically independent replicates. Benzbromarone and tolfenamic acid show synergistic effects with azithromycin/erythromycin on E. faecalis/E. faecium. All drug concentrations are in μM.

[0182] FIG. 11 shows that the antidotes dicumarol and benzbromarone selectively rescue growth of commensal bacteria from erythromycin treatment, while pathogenic enterococci are inhibited (Example 7). Heat maps show median area under the growth curve (AUC) of the seven-member synthetic community including the pathogen E. faecalis. The numbers within the heat map represent the total number of all E. faecalis viable cells for each erythromycin-antidote combination as determined by enumerating of E. faecalis colony forming units after plating on selective medium. All drug concentrations are in μM. n. d.=not detectable.

EXAMPLES

[0183] Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the description, figures and tables set out herein. Such examples of the methods, uses and other aspects of the present invention are representative only, and should not be taken to limit the scope of the present invention to only such representative examples.

[0184] Growth conditions: All experiments from this study were performed in an anaerobic chamber (Coy Laboratory Products Inc) (2% H.sub.2, 12% CO.sub.2, 86% N.sub.2) and all materials and solutions used for these experiments were pre-reduced for at least 24 h before use unless specified otherwise. Bacteria used in this study were typically pre-cultured for 2 overnights: Cells were cultured in 5 ml modified Gifu Anaerobic Medium broth (MGAM) (HyServe GmbH & Co. KG, Germany, produced by Nissui Pharmaceuticals) and grown at 37° C. overnight. The next day, cells were diluted 1/100 in 5 ml MGAM medium and grown at 37° C. for a second overnight before starting the experiments.

[0185] Quantitative assay for minimum inhibitory concentration determination with MICs test strips: MICs test strips were purchased from Liofilchem or Oxoid. All MICs were measured under anaerobic growth conditions inside a Coy anaerobic chamber. Bacteria were precultured in MGAM for two overnights and cultures were diluted to OD.sub.578=0.5.50 μl of the diluted culture were spread on a MGAM agar plate and allowed to dry for 15 min. The MIC test strip was placed on the agar with sterile tweezers, allowing the part with the lowest concentration touch the agar first. Plates were incubated at 37° C. inside the anaerobic chamber, at least overnight and longer depending on the species-specific growth requirements. After formation of a symmetrical inhibition ellipse, plates were taken out of the chamber and imaged under controlled lighting conditions (spImager S&P Robotics Inc.) using a 18 megapixel Canon Rebel T3i (Canon Inc. USA). MICs are directly determined from the strip scale at the point where the edge of the inhibition ellipse intersects the MIC test strip. All MICs were determined in duplicates. In cases of an eight-fold difference between the two values, a third replicate was done. In all cases, this resulted in a clear outlier (>8-fold different from other two MICs) that was removed from the dataset.

[0186] MIC comparison to ChEMBL and EUCAST databases: Previously known MICs were extracted from the ChEMBL database (version 24) and EUCAST (obtained on May 14, 2018). Antibiotics from these two datasets were mapped to the inventor's dataset by name. Species were mapped using NCBI Taxonomy Identifiers and species names. For MICs from ChEMBL, a keyword-based approach was used to exclude experiments on species with mutations, deletions, insertions, etc. The EUCAST database contains a large number of reported MICs for each compound-species pair. The inventors collapsed these to a single value by calculating the median MIC.

[0187] Estimates on the abundance and prevalence of species in the healthy human gut microbiome were calculated using mOTUs v2 as follows: Relative species abundances were determined in 727 shotgun metagenomic samples from donors in the control groups of multiple studies from various countries and continents. Prior to taxonomic profiling, metagenomes were quality controlled using the MOCAT2-rtf procedure, which removed reads with 95% sequence identity and an alignment length of ≥45 bp to the human genome hg19. Taxonomic profiles were then created using mOTUs version 2.1.0 with parameters -l 75; -g 2; and -c. Afterwards relative abundances below 10.sup.−4 were set to zero and species with nonzero abundance in <5 samples discarded. For the retained 1,350 species, prevalence was defined as the percentage of samples with nonzero abundance; a prevalence cut-off of 1% was chosen to classify species into “rare” and “common” species. For all species in the MIC dataset, the inventors manually assessed their status as pathogenic or non-pathogenic species using encyclopaedic and literature knowledge. Pathogenic species that occur in more than 1% of healthy people (i.e. are designated as “common”) were classified as “potentially pathogenic species” that can, for example, cause diseases in immunocompromised patients.

[0188] Killing curves and survival assay: Cells were precultured as described in the growth conditions section before being diluted to an OD.sub.578=0.01 and grown for 2 h at 37° C. (unless specified otherwise). Next, cells were diluted 1/2 in MGAM containing a 10-fold MIC of erythromycin, azithromycin or doxycycline (final antibiotic concentration is 5-fold MIC) and incubated in the presence of the antibiotic for 5 h at 37° C. At several time-points (0, 1 h, 2 h, 3 h, 4 h, 5 h), 100 μl of cells were serial-diluted in PBS (10.sup.−1 to 10.sup.−8 dilutions) and plated on MGAM-Agar plates for colony forming unit (CFU) counting. When no cells were detected using this method, a bigger volume of culture (up to 2 ml) was plated to be able to detect CFUs. Agar plates were incubated overnight at 37° C. and colonies were counted the next day, either manually, for low CFU numbers, or using the Analyze Particles tool from ImageJ.sup.35.

[0189] Live/dead staining: Cells were precultured as described in the growth conditions section before being diluted to an OD.sub.578=0.01 and grown for 2 h at 37° C. Cells were next diluted 1/2 in MGAM containing 10-fold MIC of erythromycin, azithromycin or doxycycline (final concentration is 5-fold the MIC) and incubated in the presence of the antibiotic for 5 hours at 37° C. Then, cells were live/dead stained using the LIVE/DEAD BacLight Bacterial viability and counting kit (#L34856 Molecular Probes, ThermoFisher) according to the manufacturer's protocol before and after antibiotic treatment.

[0190] Flow cytometry: Stained cells were counted using a BD LSRFortessa™ flow cytometer. The forward and side scatter signals (488 nm) as well as the green and red fluorescent signals (488-530/30 A filter and 561-610/20 A filter, respectively) were acquired. The FSC/SSC detectors were set to logarithmic scale. The flow rate varied between 12 μl/min and 60 μl/min depending on the concentration of each sample, and the analysis was stopped when 10,000 target events were measured. Graphs were generated using the FlowJo V10.3 software (Treestar).

[0191] Microscopy: For live/dead imaging, stained cells were washed twice in 0.85% NaCl before being spotted on 0.85% NaCl+1% agarose pads between a glass slide and a coverslip. For time-lapse imaging, cells were precultured as described in the growth conditions section. Cells were then diluted to an OD.sub.578=0.01 and grown for 3 h at 37° C. before being spotted on MGAM+1% agarose pads, supplemented or not with 15 μg/ml erythromycin (5-fold MIC) between a glass slide and a coverslip. Slides were sealed with valap and taken outside of the chamber for imaging. The imaging was performed using a Nikon Eclipse Ti inverted microscope, equipped with a Nikon DS-Qi2 camera, a Nikon Plan Apo Lambda 60× oil Ph3 DM phase contrast objective and a Nikon HC mCherry filter set (Ex 562/40; DM 593; BA 641/75) to detect propidium iodide fluorescence. Images were acquired with the NIS-Elements AR4.50.00 software and processed with Fiji v.2.0.0-rc-68/1.52 h.

[0192] Growth curves: Cells were precultured as described in the growth conditions section. Then, cells were diluted to an OD.sub.578=0.01 in a 96-well plate sealed with a breathable membrane (Breathe-Easy®) and grown for 2 h. Next, erythromycin was added to the culture to a final concentration of 15 μg/ml (5-fold MIC) and growth curves were acquired for 20 h using a microplate spectrophotometer (EON, Biotek) by measuring the OD.sub.578 every hour after 30 sec of linear shaking.

[0193] The examples show:

Example 1: Antibacterial Effects on Gut Microbiota

[0194] The standard way to determine whether an antibiotic has bactericidal or bacteriostatic activity is to calculate time-kill curves, where the bacterial survivors are counted on agar at various time-points after drug treatment. If, over a significant period of antibiotic treatment (ranging from a 5 to 24 hours), the number of colony forming units (CFU)/ml of culture decreases by more than 99.9%, the antibiotic is considered bactericidal.

[0195] The inventors assessed the survival of 12 abundant gut microbes over a 5-hour treatment of either a macrolide (erythromycin or azithromycin) or a tetracycline (doxycycline) at 5×MIC (FIG. 1 a+b, FIG. 2).

[0196] Surprisingly, about half of the tested species decreased in survival by >99.9%, confirming that these drugs are bactericidal to several abundant gut microbes. To confirm this further, the inventors tested the viability of B. vulgatus and E. coli ED1a upon erythromycin, azithromycin or doxycycline treatments using live/dead staining. Microscopy and flow cytometry assessment of live/dead bacteria corroborated the initial observations (FIG. 1 c). As tetracylines are considered bona-fide bacteriostatic drugs in E. coli, the inventors were surprised to see that doxycycline effectively killed the commensal E. coli ED1a (FIG. 1 a).

[0197] The inventors also noticed that B. vulgatus and B. uniformis cultures decreased density in the presence of erythromycin (FIG. 1 d). The inventors confirmed by time-lapse microscopy that this was due to lysis. Erythromycin caused cell shape defects, including blebbing, cytoplasmic shrinkage, and ultimately cell lysis in both B. vulgatus and B. uniformis (FIG. 1 e). Altogether, the differential bactericidal activity of macrolides and tetracyclines on gut commensals could provide an explanation for the strong effects these drug classes have on the gut microbiota composition of human individuals.

Example 2: Screen for Microbiome-Protective Antibiotic Antagonism

[0198] The inventors reasoned that it should be possible to identify drugs that selectively antagonize the effect of antibiotics on gut microbes, while retaining activity against pathogens. Therefore, the inventors screened the Prestwick library to identify antagonizing compounds to erythromycin or doxycycline on two abundant and prevalent gut microbes, B. vulgatus and B. uniformis.

[0199] Preparation of screening plates. The Prestwick Chemical Library was purchased from Prestwick Chemical Inc. and drugs were re-arrayed, diluted and stored in 96 well format as described before. The inventors prepared drug plates (2×drug concentration) in MGAM medium and stored them at −30° C. For each experiment, drug plates were thawed, supplemented with the respective antibiotic solution (freshly prepared in MGAM) and pre-reduced in the anaerobic chamber overnight. All rearranging and aliquoting steps were done using the Biomek FXP (Beckman Coulter) system.

[0200] Inoculation and screening conditions. Strains were grown twice overnight, the second overnight culture was diluted in MGAM to reach OD.sub.578 nm 0.04 (4×the desired starting OD). 25 μl of the diluted cultures were used to inoculate wells containing 50 μl of 2× concentrated Prestwick drug and 25 μl of the 4× concentrated antibiotic using the semi-automated, 96-well multi-channel pipette epMotion 96 (Eppendorf). Each well contained 1% DMSO, 20 μM of the Prestwick drug and a species-specific antibiotic concentration that was just inhibitory for the respective strain (0.625 μM for erythromycin, 0.04 μM doxycycline for B. uniformis and 0.08 μM doxycycline for B. vulgatus). Plates were sealed with breathable membranes (Breathe-Easy®) and OD.sub.578 was measured hourly after 30 sec of linear shaking with a microplate spectrophotometer (EON, Biotek) and an automated microplate stacker (Biostack 4, Biotek) fitted inside a custom-made incubator (EMBL Mechanical Workshop). Growth curves were collected up to 24 h. For each antibiotic, each species was screen in biological duplicates. All experiments included control wells of unperturbed growth (32 wells per run) and control wells for growth in the presence of the antibiotic only (8 wells per plate).

[0201] Analysis pipeline and hit calling: All growth curves within a plate were truncated at the transition time from exponential to stationary phase and converted to normalized AUCs using in-run control wells (no drug) as described before. The inventors then calculated z-scores based on these normalized AUCs, removed replicates with 8-fold differences in z-scores to eliminate noise effects, computed mean z scores across the two replicates and selected combinations with mean z-scores >3. This selection included 19 potential antibiotic antagonists and the inventors followed up on 14 of them (7 potential erythromycin and 7 potential doxycycline antagonists in either B. vulgatus or B. uniformis—see FIG. 4) in independent experiments.

[0202] Validation of microbiome-protective antagonists: In a first step, the inventors kept the erythromycin/doxycycline concentration constant (0.625 μM for erythromycin, 0.078 μM (B. vulgates)/0.039 μM (B. uniformis) for doxycycline) and tested concentration gradients of the potential antagonists with ranges depending on the antagonist's solubility. Compounds were purchased from independent vendors and dissolved at 100× starting concentration in DMSO. Eight 2-fold serial dilutions were prepared in 96-well plates with each row containing a different antagonist, sufficient control DMSO wells and wells with just the respective antibiotic (‘antibiotic-only’ control). These master plates were diluted in MGAM medium (50 μl) to 2×assay concentration and 25 μl freshly prepared antibiotic solution (4× test concentration) was added. Plates were pre-reduced overnight in an anaerobic chamber and inoculated with 25 μl of overnight cultures (prepared as described under “Growth conditions”) to reach a starting OD.sub.578 of 0.01 and 1% DMSO concentration. Growth was monitored hourly for 24 h after 30 sec of linear shaking. Experiments were performed in biological triplicates. For analysis, growth curves were converted into normalized AUCs (see above). The inventors accounted for residual growth in the presence of the antibiotic by subtracting the median normalized AUCs of the ‘antibiotic-only’ control per plate. The inventors computed medians across triplicates and considered a normalized AUC >0.25 as concentration-dependent growth rescue by the antagonist.

[0203] Checkerboard assays for anaerobic commensals: Validated antagonists were further investigated in 8×8 checkerboard assays, where both antibiotics and antagonists were titrated against each other. Such assays were first performed for the commensals that were originally screened (i. e. B. vulgatus and B. uniformis—4 replicates) and later expanded towards 6 further gut microbes (B. caccae, B. fragilis NT, B. ovatus, B. thetaiotaomicron, P. copri, P. distasonis—2 replicates). For vertical gradients, 2-fold serial dilutions of the antagonists were prepared first in 100× in DMSO and diluted in MGAM as described above (section ‘Validation of microbiome-protective antagonists’). Horizontal antibiotic dilution series were freshly prepared in MGAM at 4× final concentration in equidistant concentration steps. Both, vertical and horizontal dilution series were combined (50 μl of the antagonist gradients (2×) and 25 μl of the antibiotic gradients (4×)) in 96 well plates. Plates were pre-reduced under anaerobic conditions overnight, inoculated with 25 μl of diluted overnight culture (at 4× starting OD) and sealed with breathable membrane (Breathe-Easy®). Bacterial growth was monitored once per hour for 24 hours after 30 sec linear shaking (Eon+Biostack 4, Biotek) under anaerobic conditions.

[0204] Growth curves were converted into normalized AUCs as described using in-plate controls to define unperturbed growth.

[0205] Checkerboard assays for pathogens under aerobic conditions: For three pathogens (S. aureus Newman ATCC 13420, S. aureus subsp. aureus Rosenbach 1884 DSM20231 and E. faecium ATCC19434) 8×8 checkerboard assays were performed in transparent 384 well plates (Greiner BioOne GmbH), with each well containing 30 μl (50 μl for S. pneumoniae) in total. S. aureus strains were grown in tryptic soy broth, E. faecium in BHI medium. Antagonistic drug concentrations were 2-fold distant. Cell were inoculated at initial OD.sub.595 nm˜0.01 from an overnight culture. Plates were sealed with breathable membranes (Breathe-Easy), incubated at 37° C. (Cytomat 2, Thermo Scientific) with continuous shaking and OD.sub.595 nm was measured every 30 minutes for 16 hours in a Filtermax F5 multimode plate reader (Molecular Devices). Growth was evaluated at the transition to stationary phase (9 hours for S. aureus, 12 hours for E. faecium). All experiments were done at least in 2 biological replicates and 2 technical replicates. For S. pneumoniae D39, the inventors only tested concentration gradients of the potential antagonists in constant concentration of the antibiotics in BHI medium.

TABLE-US-00001 TABLE 1 Screen for microbiome-protective antagonists to erythromycin/tetracycline Mean Drug Antibiotic Strain z-score Validation Hexestrol Erythromycin B. uniformis 9.630 TRUE Diethylstilbestrol Erythromycin B. uniformis 9.258 TRUE Famprofazone Erythromycin B. uniformis 7.832 FALSE Tolfenamic acid Erythromycin B. vulgatus 7.381 TRUE Dicumarol Erythromycin B. vulgatus 7.313 TRUE Lorglumide Erythromycin B. uniformis 6.106 TRUE sodium salt Benzbromarone Erythromycin B. uniformis 6.034 FALSE Iopanoic acid Doxycycline B. vulgatus 5.698 TRUE Tiratricol, 3,3′,5- Doxycycline B. uniformis 4.333 TRUE triiodothyroacetic acid Tolfenamic acid Doxycycline B. vulgatus 4.000 TRUE Losartan Doxycycline B. uniformis 3.421 FALSE Diethylstilbestrol Doxycycline B. vulgatus 3.296 NA Celecoxib Doxycycline B. uniformis 3.275 FALSE Diflunisal Doxycycline B. vulgatus 3.265 TRUE Efavirenz Doxycycline B. vulgatus 3.138 NA Diclazuril Doxycycline B. vulgatus 3.117 NA Triclosan Doxycycline B. uniformis 3.066 FALSE Clonixin Doxycycline B. vulgatus 3.061 NA Lysinate Meclofenamic acid Doxycycline B. vulgatus 3.010 NA sodium salt monohydrate Abbreviation: NA = not available

Example 3: Validation of Microbiome-Protective Antibiotic Antagonisms

[0206] Of the 19 identified hits (FIG. 3 b, Table 1), the inventors tested the 14 candidates with the strongest activity in a concentration-dependent manner (FIG. 5 a). Nine retained antagonistic effects over a broader concentration range, which the inventors confirmed by checkerboard assays (FIG. 3 c). The antidotes that showed the strongest antagonisms were the anticoagulant drug dicumarol, and two non-steroidal anti-inflammatory drugs, tolfenamic acid and diflusinal. While dicumarol rescued B. vulgatus from erythromycin and diflusinal from doxycycline, tolfenamic acid was able to protect B. vulgatus from both drugs.

[0207] In addition, these interactions were able to at least partially rescue the killing of B. vulgatus by erythromycin and doxycycline (FIG. 5 b). The inventors then probed these 3 drugs for their ability to protect other abundant gut commensals and confirmed that both dicumarol and tolfenamic acid were able to counteract erythromycin on several species (FIG. 3 d, FIG. 6). In contrast, both drugs did not affect the potency of erythromycin on Staphylococcus aureus and Streptococcus pneumoniae, pathogens against which erythromycin is routinely prescribed (FIG. 3 e, FIG. 7 a). For example, tolfenamic acid and dicumarol at concentration ranges of 5-40 μm could rescue the growth of 5/7 tested abundant gut commensal species at clinically relevant erythromycin concentrations (FIG. 3 f, FIG. 7 b). Altogether, the inventor's data provides antidotes that specifically mask the collateral damage of antibiotics on commensals.

Example 4: Representative Species of the Order Bacteroidales can be Protected from the Exemplary Macrolide Antibiotic Azithromycin by Dicumarol, Benzbromarone or Tolfenamic Acid

[0208] The inventors followed up on earlier findings, where it was shown that the marketed drugs dicumarol, benzbromarone and tolfenamic acid can rescue the growth of B. vulgatus in the presence of erythromycin. In particular, they aimed at testing, whether these antidotes can also protect from other macrolides such as azithromycin, a clinically more relevant antibiotic. It was generally found that all antidotes as tested worked at least equally well, in some cases even better, in antagonizing azithromycin compared to erythromycin (see FIG. 8).

Example 5: Synthetic Gut Commensal Communities can be Protected from Macrolide Antibiotics by the Antidotes Dicumarol, Benzbromarone or Tolfenamic Acid

[0209] So far, antidote effects have only been analyzed in single or pure cultures. However, the human gut microbiome is a complex community of many different bacterial taxa that is usually not adequately mimicked in monocultures. Hence, the inventors created a synthetic gut commensal community consisting of B. vulgatus, B. fragilis nt, B. thetaiotaomicron, P. copri, B. caccae, B. ovatus, P. distasonis, and tested antidote effects in this setting. Growth of the community is rescued from both erythromycin and azithromycin with all three antidotes as tested, as was observed in pure culture, the rescue from azithromycin is stronger than from erythromycin (see FIG. 9).

Example 6: Human Intestinal Pathogens of the Genus Enterococcus are not Rescued from Macrolide Antibiotics by the Antidotes Dicumarol, Benzbromarone or Tolfenamic Acid

[0210] Here, the inventors expanded testing towards additional antidotes, and included the intestinal human pathogen Enterococcus faecalis. Neither dicumarol nor benzbromarone or tolfenamic acid could rescue E. faecalis/E. faecium from erythromycin or azithromycin treatment. Moreover, the antidote benzbromarone and tolfenamic acid synergized with the antibacterial effects of erythromycin/azithromycin against E. faecalis/E. faecium (see FIG. 10).

Example 7: In Enterococci-Containing Synthetic Communities, the Antidotes Dicumarol and Benzbromarone Selectively Rescue Growth of Commensal Bacteria from Erythromycin Treatment, while Pathogenic Enterococci are Inhibited

[0211] The inventors exposed synthetic communities that contained the intestinal gut pathogen E. faecalis to erythromycin, and evaluated rescue of gut commensals and simultaneous killing of the pathogen in the presence of antidotes. For both, benzbromarone and dicumarol, the inventors could show rescue of the community despite erythromycin treatment, while E. faecalis cells were efficiently inhibited. This result shows that within microbial communities, the antidotes can selectively rescue commensals from erythromycin (see FIG. 11). As an example how to interpret the plots in Figure ii, if focused on an erythromycin concentration of 1.15 μM, the community as analyzed is partially inhibited, as the tiles of the heat map indicate in pale shading. The community still contains ˜9×10.sup.5 E. faecalis cells. If then 40 μM dicumarol or 20 μM benzbromarone is added, the community is rescued, as the AUCs are higher/tiles are filled in darker shading color. However, E. faecalis counts drop/are still found at below the detection limit. Thus, only non-pathogenic community members are rescued in this assay.