WO2024003042A1

WO2024003042A1 - Novel combinations of sesquiterpene alcohols, polymyxins and nisin compounds as antimicobial and/or antifungal compounds

Info

Publication number: WO2024003042A1
Application number: PCT/EP2023/067458
Authority: WO
Inventors: Jesper Pedersen
Original assignee: Breakthrough Biotech Drift Aps
Priority date: 2022-06-28
Filing date: 2023-06-27
Publication date: 2024-01-04

Abstract

The present invention relates to a composition comprising the combination of at least the following three components one or more sesquiterpene alcohol compound; one or more polymyxin compound or a derivative thereof; and one or more anti-microbial compound.

Description

NOVEL COMBINATIONS OF SESQUITERPENE ALCOHOLS, POLYMYXINS AND NISIN COMPOUNDS AS ANTIMICOBIAL AND/OR ANTIFUNGAL COMPOUNDS

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a novel combination of antimicrobial and/or antifungal compounds with low cytotoxic potential for the inhibition of fungal and/or bacterial growth and for the treatment of fungi and/or bacterial infections.

BACKGROUND OF THE INVENTION

The increasing incidence of microbial resistance to antibiotics and other antimicrobials is a growing concern facing the medical, food and sanitation industries. In 2015, antibioticresistant bacteria of public health concern in countries of the EU and European Economic Area (EEA), measured in a number of cases, attributable deaths, and disability-adjusted life-years (DALYs) and reported that 671,689 cases of infections with selected antibioticresistant bacteria occurred in 2015 in the EU and EEA and that this lead to a mortality of 644 deaths per 100.000 population.

At the same time, multi-resistant fungi are a well-known problem for global health too. One example is Candida auris, which has been identified globally, as a significant nosocomial multidrug-resistant (MDR) pathogen responsible for causing invasive outbreaks.

Major mechanisms of microbial resistance may include active drug efflux systems and mutations which lead to nonspecific decreases in cell permeability. Other routes to resistance may include antimicrobial degradation, inactivation by enzymatic modifications, or alteration of the drug's target within the cell.

To counter the increasing incidence of antimicrobial resistance, pharmaceutical, academia, biotech and other pharmaceutical industries have invested substantial resources in the search for new inhibitory compounds of microbial, plant and animal origin. Newer strategies to overcome antimicrobial resistance have included increased production of new synthetic and semisynthetic antibiotics which are resistant to the activities of those microbial enzymes (i.e. ss-lactamases) capable of degradation or modification of naturally- derived antibiotics. Although many specific mechanisms of microbial resistance have been successfully addressed, it is thought that the more general mechanisms of altered permeability and increased efflux capability will become increasingly important from a clinical perspective.

Efflux systems and mechanisms of antibiotic degradation rely on saturable biological structures (i.e., pumps or enzymes). If the nonspecific influx of an intracellularly or membrane-targeted lethal agent is high enough to overcome these inactivation mechanisms, then the effect of these mechanisms might be minimized, leading to the death of the cell.

Due to the increasing challenges of finding non-resistant antimicrobial compounds and/or antifungal compounds for efficient inhibition of bacterial and/or fungal growth, alternative solutions to traditional e.g., antibiotics are in high demand. Finding new, efficient treatments can save lives and quality of life for many patients worldwide.

SUMMARY OF THE INVENTION

Thus, an object of the present invention relates to providing a novel non-resistant antimicrobial and/or antifungal compositions demonstrating efficient inhibition of bacterial and/or fungal growth.

Thus, one aspect of the present invention relates to a composition comprising a combination of at least two of the following components: one or more sesquiterpene alcohol compound; one or more polymyxin compound or a derivative thereof; and/or one or more nisin-compound.

Another aspect of the present invention relates to a composition comprising the combination of at least the following three components:

(i) one or more sesquiterpene alcohol compound;

(ii) one or more polymyxin compound or a derivative thereof; and

(iii) one or more anti-microbial compound. Another aspect of the present invention relates to a composition comprising one or more sesquiterpene alcohol compound and one or more polymyxin compound or a derivative thereof.

Yet another aspect of the present invention relates to a composition consisting essentially of a combination of at least two of the following components: one or more sesquiterpene alcohol compound; one or more polymyxin compound or a derivative thereof; and/or one or more nisin-compound.

One aspect of the present invention relates to a composition consisting essentially of a combination of one or more sesquiterpene alcohol compound and one or more polymyxin compound or a derivative thereof.

A further aspect of the present invention relates to an antibacterial product comprising the composition according to the present invention.

An even further aspect of the present invention relates to an antifungal product comprising the composition according to the present invention.

Yet a further aspect of the present invention relates to a pharmaceutical agent comprising the composition according to the present invention, for use as a medicament.

Yet an aspect of the present invention relates to a pharmaceutical agent comprising the composition according to the present invention, for use in the treatment of bacterial, and/or fungal infections in a mammal.

A further aspect of the present invention relates to inhibition and/or treatment of fungi and/or bacterial growth and/or infections by administering to a mammal a single composition comprising a mix of: one or more sesquiterpene alcohol compound; one or more polymyxin compound or a derivative thereof; and one or more anti-microbial compound; or by administering to a mammal the: one or more sesquiterpene alcohol compound; one or more polymyxin compound or a derivative thereof; and one or more anti-microbial compound; as individual or separate compounds. An even further aspect of the present invention relates to a composition comprising : one or more sesquiterpene alcohol compound; one or more polymyxin compound or a derivative thereof; and one or more anti-microbial compound; and at least one of: at least one organic solvent or at least one alcohol; and/or water or an aqueous solution; and/or a pH-value below 7.0, such as below 6.5, e.g. below 6.0, such as below 5.5, e.g. below 5.0, such as below 4.5, e.g. below 4.0, such as below 3.5, e.g. below 3.0, such as below 2.5.

An even further aspect of the present invention relates to a combinational product comprising : one or more sesquiterpene alcohol compound; one or more polymyxin compound or a derivative thereof; and one or more anti-microbial compound.

Yet an aspect of the present invention relates to a method for providing a composition according to the present invention, wherein the method comprises the steps of: a. providing one or more sesquiterpene alcohol compound; b. providing one or more polymyxin compound or a derivative thereof; and c. providing one or more anti-microbial compound; d. combining the compounds provided in steps a, b and c.

DETAILED DESCRIPTION OF THE INVENTION

Accordingly, the inventors of the present invention surprisingly found that a composition comprising a certain combination of compounds provides a synergistic effect against microbial growth. It was surprisingly found that the composition according to the present invention demonstrate a strong synergistic effect against the growth of bacterial growth as well as a strong synergistic effect against the growth of fungus.

Hence, a preferred embodiment of the present invention relates to a composition comprising the combination of at least the following three components: (i) one or more sesquiterpene alcohol compound;

(ii) one or more polymyxin compound or a derivative thereof; and

(iii) one or more anti-microbial compound.

A further preferred embodiment of the present invention relates to a composition comprising at least two of the three compounds listed below: one or more sesquiterpene alcohol compound; one or more polymyxin compound or a derivative thereof; and/or one or more nisin-compound or a derivative thereof.

Preferably, the present invention relates to a composition consisting essentially of a combination of at least two of the following components: one or more sesquiterpene alcohol compound; one or more polymyxin compound or a derivative thereof; and/or one or more nisin-compound or a derivative thereof.

The composition according to the present invention may be a mix of the three components into a single composition or a combination of the three compounds as individual or separate compounds.

To fight microorganisms invading the mammal body (e.g. human or animal body, in particular the human body) the individual compounds may traditionally be used in high concentrations to be sufficiently effective against the invading microorganism. High concentrations of the compounds may however show toxic effects to the mammal organism (e.g. human or animal organism). Thus, it is desirable to find a way to reduce the concentration or the amount of the individual compounds (the one or more sesquiterpene alcohol compound, the one or more polymyxin compound or a derivative thereof and the one or more anti-microbial compound) of the composition.

Even a reduction in the concentration or the amount of the individual compounds (the one or more sesquiterpene alcohol compound, the one or more polymyxin compound or a derivative thereof and the one or more anti-microbial compound) may be important, a synergistic effect of the composition according to the present invention may be of even further importance, since the individual compounds used in the present invention are toxic to human and/or animal cell lines when used at increased or high concentrations. By combining the compounds (the one or more sesquiterpene alcohol compound, the one or more polymyxin compound or a derivative thereof and the one or more anti-microbial compound), and even providing a synergistic effect, it may be possible to achieve inhibition of microorganisms, in particular of bacteria and fungi, when using the composition according to the present invention with individual compounds below their toxic levels, when used for humans or animals. The synergy may also allow the substitution of different amount of one compound, by adding smaller amounts of one of the other compounds to avoid toxicity.

In prior art, it has been found that the one or more polymyxin compound or a derivative thereof, e.g. PMBN, in high concentrations (e.g. at concentrations or amounts above 500 mg/ml is nephrotoxicity in ex vivo studies. Furthermore, it was found that polymyxin nonapeptide derivatives demonstrated acute toxicity and that PMBN demonstrated some toxicity against K562 cells. It has also been shown that polymyxin compounds, e.g. PMBN and polymyxin B, may accumulate in the kidneys and causes kidney damage and nephrotoxicity, though PMBN is a lot less toxic than polymyxin B and the reason it is added to the invention.

Furthermore, the one or more anti-microbial compound, in particular nisin, may have toxic effects on various human cell lines when provided in too high concentrations or amounts, e.g. at concentrations above 600 pg/ml. The toxic effect was shown on gingival fibroblasts and showed to exert a hemolytic effect, e.g. by inhibition of rabbit red blood cells were hemolysis would increase with increasing nisin concentration. Increased concentrations of nisin may also lead to sperm motility and infertility. High nisin concentrations may also provide cytotoxicity of gastrointestinal cells, like Caco-2, HT29 and HUVEC cells, as well as damages or viability to kidney epithelial Vero cells, MCF-7 and HepG2 cells.

The one or more sesquiterpene alcohol compound, in particular the farnesol compound, have also been demonstrated to have toxic effect when applied in high concentrations (e.g. concentrations above 500 pl/mg). In particular, these high concentrations of the farnesol compound have shown in rats to cause acute oral toxicity by oral gavage where clinical signs and even mortality were observed over a period of 14 days. Decreased motor activity, coordination disturbance, piloerection and diarrhea may be observed approximately 20 minutes after dosing the high concentrations. Furthermore, it was found that farnesol compounds show a toxic potential by inducing apoptosis in different types of cell lines.

The results above demonstrates that the different drugs all can have a toxic effect on humans when applied in too high concentrations. By utilizing the combination and furthermore the synergy provided by the composition according to the present invention the inventor was able to construct and optimize a new antibiotic in two ways:

1) use a different compound selected from the compounds in the invention, i.e. one or more sesquiterpene alcohol compound; one or more polymyxin compound or a derivative thereof; and one or more anti-microbial compound, if one of the compound is at a toxic (or near a toxic) concentration; and/or

2) use the synergy to reduce the overall amount of the compounds because the synergy allows the use of a composition whose total sum of masses are less than the mass necessary when only one or two of the compounds of the invention are used to inhibit a microorganism, e.g. the bacteria or fungus.

A preferred embodiment of the present invention relates to a composition comprising the combination of at least the following three components:

(i) one or more sesquiterpene alcohol compound;

(ii) one or more polymyxin compound or a derivative thereof; and

(iii) one or more anti-microbial compound.

The one or more anti-microbial compound may be a compound that kills microorganisms or stops their growth.

Preferably, the anti-microbial compound may be a compound different from the one or more sesquiterpene alcohol compound and different from the one or more polymyxin compound or a derivative thereof.

Examples of anti-microbial compound may be a nisin compounds, a penicillin, a tetracycline, a cephalosporin, a quinolone, a lincomycin, a macrolide, a sulfonamide, a glycopeptide, an aminoglycoside, or a carbapenem or an azole-compound (like ketoconazole, itraconazole, fluconazole, fosfluconazole, voriconazole, posaconazole, or isavuconazole), or a combination hereof.

Anti-microbials may be selected from the group of compounds selected from aminoglycosides, ansamycins, carbacephem, carbapenems, cephalosporins, glycopeptides, lincosamides, lipopeptide, macrollides, monobactams, nitrofurans, oxazolidinones, penicillins, polypeptides, puinolones, fluoroquinolones, Sulfonamides, tetracyclines, antimycobacterials, amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, streptomycin, spectinomycin, ansamycins, geldanamycin, herbimycin, rifaximin, carbacephem, loracarbef, carbapenems, ertapenem, doripenem, imipenem, cilastatin, meropenem, cephalosporins, cefadroxil, cefazolin, cefalotin or cefalothin, cephalexin, cefaclor, cefamandole, cefoxitin, cefprozil, cefuroxime, cefixime, cefdinir, cefditoren, cefoperaZone, cefoperaZone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cephalosporins, cefepime, ceftaroline fosamil, ceftobiprole, teicoplanin, vancomycin, telavancin, dalbavancin, oritavancin, lincosamides, clindamycin, lincomycin, daptomycin, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, tellithromycin, spiramycin, monobactams, aztreonam, nitrofurans, furazolidone, nitrofurantoin, oxazolidinone, lineZolid, posiZolid, radeZolid, toreZolid, amoxicillin, amplicillin, azlocillin, carbenicillin, cioxacillin, dicloxacillin, flucioxacillin, mezlocillin, methicillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, penicillin F, penicillin K, temocillin, ticarcillin, bacitracin, ciprofloxacin, enoxacin, gatifloxacin, gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nalidixic acid, nisin, norfloxacin, ofloxacin, trovafloxacin, grepafloxacin, sparfloxacin, temafloxacin, mafenide, Sulfacetamide, Sulfadiazine, silver Sulfadiazine, Sulfadimethoxine, Sulfamethizole, Sulfamethoxazole, Sulfanilamide, Sulfasalazine, Sulfisoxazole, Sulfonamidochrysoidine, demeclocycline, doxycycline, minocycline, oxytetracycline, tetracycline, clofazimine, dapsone, capreomycin, cycloserine, ethambutols, thionamide, isoniazid, pyrazinamide, rifampicin/rifampin, rifabutin, rifapentine, Streptomycin, arsphenamine, fosfomycin, fusidic acid, metronidazole, mupirocin, platensimycin, quinupristin/dalfopristin, thiamphenicol, tigecyclines, tinidazole, trimethoprims, and a combination hereof.

Preferably, the anti-microbial compound may comprise a peptide, preferably an antimicrobial peptide compound.

In an embodiment of the present invention the anti-microbial compound may be selected from the group of anti-microbial compounds selected from a subtilin polypeptide fragment, Ericin S, Geobacillin, an epidermin polypeptide fragment, a (HV 16L) epidermin polypeptide fragment, a mutacin B-Ny266 polypeptide fragment, a mutacin 1140 polypeptide fragment, a component with binding interactions with a pyrophosphate of bacterial cell wall precursor Lipid II.

Preferably, the anti-microbial compound according to the present invention may be one or more nisin-compound or a derivative thereof. Preferably, the anti-microbial compound may be one or more nisin-compound. The nisin-compound or a derivative thereof may relate to a biologically active equivalent having substantially equivalent function when compared to the corresponding wild-type nisin at 37°C. In an embodiment of the invention a nisin-compound or a derivative thereof has about 1, 2, 3, 4, or 5 or less conserved amino acid substitutions to the wild type nisin.

The nisin-compound or a derivative thereof exposed to varying conditions (e.g., different buffers, temperature, different additives, etc.) may have 80, 85, 90, 95% or more biological activity of a wild-type nisin at 37°C., in phosphate buffer.

The nisin-compound or a derivative thereof may be part of the invention and are at least about 80%, or about 81, 82, 83, 84,85, 86, 87, 88, 89, 90,91,92,93, 94.95,96.97,98, or 99% identical to any of the nisin-components disclosed herein. For example, a nisin- compound or a derivative thereof may have about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid changes. In one embodiment of the invention a polypeptide has about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or less conserved amino acid substitutions.

In an embodiment of the present invention the nisin-compound or a derivative thereof may be part of a fusion protein, which can contain heterologous amino acid sequences. Heterologous amino acid sequences can be present at the C or N terminus of a nisin- compound or a derivative thereof to form a fusion protein. More than one the nisin- compound or a derivative thereof may be present in a fusion protein. Fragments of the nisin-compound or a derivative thereof may be present in a fusion protein of the invention. A fusion protein of the invention can comprise one or more nisin-compound or a derivative thereof, fragments thereof, or combinations thereof.

In an embodiment of the present invention the one or more nisin-compound may be a nisin variant A, a nisin variant Z, a nisin variant F, a nisin variant Q, a nisin variant H, a nisin variant J, a nisin variant P, a nisin variant U, a derivative constructed by substituting one or more amino acids, a peptide with a similar molecular structure, or any combination hereof. Preferably, the one or more nisin compound may be a nisin variant A or a nisin variant Z, even more preferably the one or more nisin compound may be a nisin variant Z. In a further embodiment of the present invention the anti-microbial compound may be a combination of one or more nisin compounds in combination with one or more antimicrobial compound selected from amoxicillin, amplicillin, azlocillin, carbenicillin, cioxacillin, dicloxacillin, flucioxacillin, mezlocillin, methicillin, nafcillin, oxacillin, penicillin G (benzylpenicillin), penicillin V (phenoxymethylpenicillin), piperacillin, penicillin F (2- pentenylpenicillin), penicillin K (n-heptylpenicillin), penicillin X (p-hydroxybenzylpenicillin) temocillin or tica rcillin . In the context of the present invention, a derivative is a compound that is derived from the original compound by a chemical reaction or a compound that can be imagined arising from the original compound, such as if one atom is replaced with another atom or group of atoms; or if one amino acid is replaced with another amino acid or group of amino acids.

Further the context of the present invention, the term "consists essentially of", relates to a limitation of the scope of a claim to the features or steps specified, and to those features or steps, not mentioned and that do not materially affect the basic and novel characteristic(s) of the claimed invention.

Preferably, the one or more sesquiterpene alcohol compound may be a farnesol compound.

The present invention further provides a composition wherein the one or more polymyxin compound may be a polymyxin B compound, even more preferably the one or more polymyxin may be a polymyxin B nonapeptide (PMBN).

In an embodiment of the present invention the composition may comprise (or consist essentially of) one or more sesquiterpene alcohol compound or a derivative thereof and one or more polymyxin or a derivative thereof.

The inventor of the present invention surprisingly found that a composition comprising the combination of one or more sesquiterpene alcohol compound and one or more polymyxin compound provided a synergistic effect in sensitizing the microbial cells and improving the effects of anti-microbial compounds.

In another embodiment of the present invention the composition may comprise (or consist essentially of) one or more sesquiterpene alcohol or a derivative thereof compound and one or more nisin-compound or a derivative thereof.

In a further embodiment of the present invention the composition may comprise (or consist essentially of) one or more polymyxin or a derivative thereof and one or more nisin-compound or a derivative thereof.

In yet an embodiment of the present invention the composition comprises one or more sesquiterpene alcohol compound or a derivative thereof, one or more polymyxin or a derivative thereof and one or more nisin-compound or a derivative thereof.

Preferably, the composition according to the present invention comprises one or more polymyxin compounds, wherein the one or more polymyxin compound is PMBN in combination with one of more sesquiterpene alcohol compound or a derivative thereof (in particular farnesol) and/or one or more nisin-compound or a derivative thereof (in particular nisin variant A or a nisin variant Z, preferably a nisin variant Z).

It is believed that one or more sesquiterpene alcohol compound (e.g. farnesol) and the one or more polymyxin compound or a derivative thereof has a function of permeabilizing the cell wall allowing access or increased access of the one or more anti-microbial compound to kill the microorganism.

To further improve the effect of the present invention other types of permeabilizers may be added. Examples of such other permeabilizers may include chelating agents like EDTA or agents that act directly on the cell membrane of the microorganism, affecting permeability and leading to leakage of the intracellular compounds, such as detergents, cationic peptides, gramicidin A, magainins, defensins, daptomycin, lantibiotics which have a permeabilising effect on the membrane or other molecules with a permeabilising effect on the membrane.

Optionally, the composition according to the present invention may also be combined with one or more antimicrobials, antibiotics, bacteriocins, anti-viral, Virucidal, or anti-fungal compounds or molecules to form a further improved composition.

Preferably, the composition according to the present invention comprising one or more sesquiterpene alcohol compound, in particular the farnesol compound or a derivative thereof, in the composition in the range of 0.001-600 pg/ml, such as in the range of 0.005-450 pg/ml, e.g. in the range of 0.01-300 pg/ml, such as in the range of 0.025-150 pg/ml, e.g. in the range of 0.05-75 pg/ml, such as in the range of 0.075-25 pg/ml, e.g. in the range of 0.1-10 pg/ml, such as in the range of 0.15-5 pg/ml, e.g. in the range of 0.2-2 pg/ml, such as in the range of 0.25-1 pg/ml, e.g. in the range of 0.3-0.75 pg/ml, such as in the range of 0.035-0.5 pg/ml, e.g. in the range of 0.01-0.1 pg/ml, such as in the range of 0.025-0.05 pg/ml.

The composition according to the present invention comprising one or more polymyxin compound or a derivative thereof, in particular polymyxin B nonapeptide (PMBN), in the composition in the range of 0.001-600 pg/ml, such as in the range of 0.005-450 pg/ml, e.g. in the range of 0.01-300 pg/ml, such as in the range of 0.025-150 pg/ml, e.g. in the range of 0.05-75 pg/ml, such as in the range of 0.075-25 pg/ml, e.g. in the range of 0.1- 10 pg/ml, such as in the range of 0.15-5 pg/ml, e.g. in the range of 0.2-2 pg/ml, such as in the range of 0.25-1 pg/ml, e.g. in the range of 0.3-0.75 pg/ml, such as in the range of 0.035-0.5 pg/ml, e.g. in the range of 0.01-0.1 pg/ml, such as in the range of 0.025-0.05 pg/i-nl.

In an embodiment of the present invention the composition comprising one or more nisin- compound, in particular nisin variant Z, in the composition in the range of 0.001-600 pg/ml, such as in the range of 0.005-200 pg/ml, e.g. in the range of 0.01-100 pg/ml, such as in the range of 0.025-50 pg/ml, e.g. in the range of 0.05-75 pg/ml, such as in the range of 0.075-25 pg/ml, e.g. in the range of 0.1-10 pg/ml, such as in the range of 0.15-5 pg/ml, e.g. in the range of 0.2-2 pg/ml, such as in the range of 0.25-1 pg/ml, e.g. in the range of 0.3-0.75 pg/ml, such as in the range of 0.035-0.5 pg/ml, e.g. in the range of 0.01-0.1 pg/ml, such as in the range of 0.025-0.05 pg/ml.

Preferably, the present invention provides a composition comprising one or more sesquiterpene alcohol compound or a derivative thereof, in particular the farnesol compound, and/or one or more polymyxin compound or a derivative thereof, in particular polymyxin B nonapeptide, and/or one or more nisin-compound or a derivative thereof, in particular nisin variant Z, wherein all compounds are in any of the above-mentioned ranges.

In an embodiment the composition according to the present invention may consist essentially of a combination of farnesol at a farnesol-content in the entire composition in the range of lx IO^-7 - 6x l0^-2 % (w/w), e.g. in the range of lx IO^-7 - 3x l0^-3 % (w/w), such as in the range of Ix lO^-6 - Ix lO^-4 % (w/w), e.g. in the range of 5xl0^-5 - Ix lO^-5 % (w/w), in combination with one or both of PMBN (at a PMBN-content in the entire composition in the range of Ix lO^-7 - 6x l0^-2 % (w/w), e.g. in the range of Ix lO^-7 - 3x l0^-3 % (w/w)) such as in the range of Ix lO^-6 - IxlO^-4 % (w/w), e.g. in the range of 5xl0^-5 - Ix lO^-5 % (w/w), and/or nisin Z, preferably at a nisin Z-content in the entire composition in the range of Ix lO^-7 - 6x l0^-2 % (w/w), e.g. in the range of Ix lO^-7 - 3x l0^-3 % (w/w)) such as in the range of Ix lO^-7 - Ix lO^-4 % (w/w), e.g. in the range of Ix lO^-6 - Ix lO^-5 % (w/w).

In a further embodiment the composition according to the present invention may comprise one or more sesquiterpene alcohol compound or a derivative thereof, in particular the farnesol compound, and one or more polymyxin compound or a derivative thereof, in particular polymyxin B nonapeptide, wherein the compounds are provided in a ratio in the range of 1-5: 1-5, such as in the range of 1-3: 1-3; e.g. in the range of 1-2: 1-2; such as a ratio of about 1 : 1. Another embodiment of the present invention provides a composition comprising one or more sesquiterpene alcohol compound or a derivative thereof, in particular the farnesol compound, and one or more polymyxin compound or a derivative thereof, in particular polymyxin B nonapeptide wherein the compounds are with a ratio about 1 : 1; 1 :2; 1 :3, 1:4; 1 :5; 2: 1; 2:3; 2:5; 3: 1; 3:2; 3:4; 3:5; 4: 1; 4:3; 4:5; 5: 1; 5:2; 5:3; 5:4. The one or more sesquiterpene alcohol compound or a derivative thereof, in particular the farnesol compound, and one or more polymyxin compound or a derivative thereof, in particular polymyxin B nonapeptide may even be provided in a ratio about in 1 : 10, 1 :20, 1 :50, 10: 1, 20: 1, 50: 1, respectively, such as in a ratio in the range of 1 :50- 50: 1; e.g. in the range of 1 :20-20: 1; such as in the range of 1 : 10-10: 1; e.g. in the range of 1 :5-5: 1, such as in the range of 1 :3-3: 1, e.g. in the range of 1 :2-2: 1.

In a further embodiment of the present invention the composition may comprise one or more sesquiterpene alcohol compound or a derivative thereof, in particular the farnesol compound, and one or more polymyxin compound or a derivative thereof, in particular polymyxin B nonapeptide, wherein the ratio of one or more sesquiterpene alcohol compound or a derivative thereof may be higher than the content of one or more polymyxin compound or a derivative thereof, in particular polymyxin B nonapeptide.

In yet an embodiment of the present invention the ratio between the one or more sesquiterpene alcohol compound or a derivative thereof, in particular the farnesol compound, and the one or more polymyxin compound or a derivative thereof, in particular polymyxin B nonapeptide may be at least 10: 1, such as at least 20: 1, e.g. at least 50: 1, such as at least 100: 1, e.g. at least 250: 1, such as at least 500: 1, e.g. at least 750: 1, such as at least 1000: 1, e.g. at least 2000: 1, such as in the range of 10: 1 to 5000: 1, e.g. in the range of 20: 1 to 2500: 1, such as in the range of 50: 1 to 2000: 1, e.g. in the range of 100: 1 to 1000: 1, such as in the range of 750: 1 to 250: 1.

In a further embodiment of the present invention the ratio between the one or more polymyxin compound or a derivative thereof, in particular polymyxin B nonapeptide and the one or more sesquiterpene alcohol compound or a derivative thereof, in particular the farnesol compound, may be at least 10: 1, such as at least 20: 1, e.g. at least 50: 1, such as at least 100: 1, e.g. at least 250: 1, such as at least 500: 1, e.g. at least 750: 1, such as at least 1000: 1, e.g. at least 2000: 1, such as in the range of 10: 1 to 5000: 1, e.g. in the range of 20: 1 to 2500: 1, such as in the range of 50: 1 to 2000: 1, e.g. in the range of 100: 1 to 1000: 1, such as in the range of 750: 1 to 250: 1.

In yet an embodiment the composition according to the present invention may comprises one or more sesquiterpene alcohol compound or a derivative thereof, in particular the farnesol compound, and one or more nisin-compound, in particular nisin variant Z, wherein the compounds are provided in a ratio in the range of 1-5:3-30, such as in the range of 1- 3:3-10; e.g. in the range of l-2:3-5; such as a ratio of about 1 :3. Another embodiment of the present invention provides a composition comprising one or more sesquiterpene alcohol compound or a derivative thereof, in particular the farnesol compound, and one or more nisin-compound, in particular nisin variant Z, wherein the compounds are with a ratio about 1:30; 1:10; 1:5; 1:3; 1:1; 1:15; 2:5; 2:3; 3:10; 3:5; 4:30; 4:10; 4:5; 4:3; 1:6; 1:2; 5:3.

In a further embodiment of the present invention the composition may comprise one or more sesquiterpene alcohol compound or a derivative thereof, in particular the farnesol compound, and one or more nisin-compound, in particular nisin variant Z, wherein the ratio of one or more sesquiterpene alcohol compound or a derivative thereof may be lower than the content of the one or more nisin-compound, in particular nisin variant Z. Preferably, the ratio between the one or more sesquiterpene alcohol compound or a derivative thereof, in particular the farnesol compound, and the and one or more nisin- compound, in particular nisin variant Z, may be at least 1:2, such as, at least 1:5, e.g. at least 1:10, such as at least 1:20, e.g. at least 1:50, such as at least 1:100, e.g. at least 1:250, such as at least 1:500, e.g. at least 1:750, such as at least 1:1000, e.g. at least 1:1500, such as in the range of 1:10 to 1:1500, e.g. in the range of 1:20 to 1:1000, such as in the range of 1:50 to 1:750, e.g. in the range of 1:100 to 1:700, such as in the range of 250:1 to 600:1, e.g. in the range of 400:1 to 550:1.

In an embodiment the composition according to the present invention comprises one or more polymyxin compound or a derivative thereof, in particular polymyxin B nonapeptide, and one or more nisin-compound, in particular nisin variant Z, wherein both compounds are provided in a ratio in the range of 1-5:3-30, such as in the range of 1-3:3-10; e.g. in the range of l-2:3-5; such as a ratio of about 1:3. Another embodiment of the present invention provides a composition comprising one or more polymyxin compound, or a derivative thereof, in particular polymyxin B nonapeptide and one or more nisin- compound, in particular nisin variant Z, wherein both compounds are with a ratio of about 1:30; 1:10; 1:5; 1:3; 1:1; 1:15; 2:5; 2:3; 3:10; 3:5; 4:30; 4:10; 4:5; 4:3; 1:6; 1:2; 5:3.

In a further embodiment of the present invention the composition may comprise one or more nisin-compound, in particular nisin variant Z, and one or more polymyxin compound or a derivative thereof, in particular polymyxin B nonapeptide, wherein the ratio of one or more nisin-compound, in particular nisin variant Z may be higher than the content of one or more polymyxin compound or a derivative thereof, in particular polymyxin B nonapeptide. Preferably, the ratio between the one or more nisin-compound, in particular nisin variant Z, and the one or more polymyxin compound or a derivative thereof, in particular polymyxin B nonapeptide may be at least 10:1, such as at least 20:1, e.g. at least 50:1, such as at least 100:1, e.g. at least 250:1, such as at least 500:1, e.g. at least 750:1, such as at least 1000:1, e.g. at least 2000:1, such as in the range of 10:1 to 5000:1, e.g. in the range of 20: 1 to 2500:1, such as in the range of 50: 1 to 2000:1, e.g. in the range of 100:1 to 1000:1, such as in the range of 750:1 to 250:1.

In yet an embodiment of the present invention the composition comprising one or more sesquiterpene alcohol compound or a derivative thereof, in particular the farnesol compound, and one or more polymyxin compound or a derivative thereof, in particular polymyxin B nonapeptide and one or more nisin-compound or a derivative thereof, in particular nisin variant Z, wherein the compounds are in a ratio in the range of 1-5: 1-5:3- 30, such as in the range of 1-3:1-3:3-10; e.g. in the range of l-2:l-2:3-5; such as a ratio of about 1:1:3. Another embodiment of the present invention provides a composition comprising one or more sesquiterpene alcohol compound, in particular the farnesol compound, and one or more polymyxin compound or a derivative thereof, in particular polymyxin B nonapeptide, and one or more nisin-compound, in particular nisin variant Z, wherein the compounds are with a ratio of about: 1:1:3; 1:1:5; 1:1:10; 1:1:30; 1:1:50; 1:1:100; 1:1:300; 1:2:3; 1:2:5; 1:2:10; 1:2:30; 1:3:3; 1:3:5; 1:3:10; 1:3:30; 1:4:3; 1:4:5; 1:4:10; 1:4:30; 1:5:3; 1:5:5; 1:5:10; 1:5:30; 2:1:3; 2:1:5; 2:1:10; 2:1:30; 2:2:3; 2:2:5; 1:1:15; 2:3:3; 2:3:5; 2:3:10; 2:3:30; 2:4:3; 2:4:5; 1:2:15; 2:5:3; 2:5:5; 2:5:10; 2:5:30; 3:1:3; 3:1:5, 3:1:10; 3:1:30; 3:2:3; 3:2:5; 3:2:10; 3:2:30; 1:1:1; 3:3:5; 3:3:10; 3:4:3; 3:4:5; 3:4:10; 3:4:30; 3:5:3; 3:5:5; 3:5:10; 3:5:30; 4:1:3; 4:1:5; 4:1:10; 4:1:30; 4:2:3; 4:2:5; 4:2:30; 4:3:3; 4:3:5; 4:3:10; 4:3:30; 4:4:3; 4:4:5; 4:4:30; 4:5:3; 4:5:5; 4:5:10; 4:5:30; 5:1:3; 5:1:5; 5:1:10, 5:1:30; 5:2:3; 5:2:5; 5:2:10; 5:2:30; 5:3:3; 5:3:5; 5:3:10; 5:3:30; 5:4:3; 5:4:5; 5:4:10; 5:4:30; 5:5:3; 1:1:2; 1:1:6.

A further embodiment the composition according to the present invention comprising one or more sesquiterpene alcohol compound, in particular the farnesol compound, and one or more polymyxin compound ora derivative thereof, in particular polymyxin B nonapeptide, and one or more nisin-compound, in particular nisin variant Z, wherein the compounds are with a ratio in the range about 20-1000:1:20-1500, such as in the range of 250- 750:1:750-1250; e.g. in the range about 400-600:1:900-1100, such as about 500:1:1000.

The composition according to the present invention may comprise a pH-value below 7.0, such as below 6.5, e.g. below 6.0, such as below 5.5, e.g. below 5.0, such as below 4.5, e.g. below 4.0, such as below 3.5, e.g. below 3.0, such as below 2.5, e.g. in the range of pH 2.0-8.0, such as in the range of pH 3.0-7.0, e.g. in the range of pH 3.5-6.5, such as in the range of pH 4.0-6.0, e.g. in the range of pH 4.5-5.5. In one embodiment of the present invention, the composition consists essentially of farnesol in combination with one or both of PMBN and/or nisin Z. In another embodiment of the present invention, the composition consists essentially of combination of farnesol, PMBN and nisin Z.

A preferred embodiment of the present invention relates to an antibacterial and/or an antifungal product comprising a composition comprising at least two of the three compounds listed below: one or more sesquiterpene alcohol compound; one or more polymyxin compound or a derivative thereof; and/or one or more nisin-compound.

The antibacterial and/or the antifungal product according to the present invention may preferably comprise a composition as described above.

Preferably, the antibacterial product according to the present invention may inhibit proliferation of one or more gram-positive bacteria and/or gram-negative bacteria; or for the treatment, of bacterial infections caused by a gram-positive bacterium and/or a gramnegative bacterium and/or mycobacteria. Furthermore, the antibacterial product provided by the present invention can be used for treatment of bacterial infections.

The composition according to the present invention may inhibit, reduce, prevent and/or treat growth, proliferation of one or more gram-positive bacteria, and one or more gramnegative bacteria.

The composition according to the present invention may inhibit, reduce, prevent and/or treat growth, proliferation of one or more gram-positive bacteria, one or more gramnegative bacteria and one or more fungal species, preferably a pathogenic fungus species. This composition inhibiting, reducing, preventing and/or treating growth, proliferation of various bacterial and fungal species may be a combined antibacterial/antifungal product.

A preferred embodiment of the present invention relates to inhibition and/or treatment of fungi and/or bacterial growth and/or infections by administering to a mammal a single composition comprising a mix of: one or more sesquiterpene alcohol compound; one or more polymyxin compound or a derivative thereof; and one or more anti-microbial compound; or by administering to a mammal the: one or more sesquiterpene alcohol compound; one or more polymyxin compound or a derivative thereof; and one or more anti-microbial compound; as individual or separate compounds.

The individual or separate compounds may be administered separately to the mammal (as non-mixed compounds) as they may be provided to a mammal at different time intervals but may be considered a combined treatment.

In an embodiment of the present invention microbial infection may be inhibited, reduced, and/or prevented by supplying to a mammal at least the following three components:

(i) one or more sesquiterpene alcohol compound;

(ii) one or more polymyxin compound or a derivative thereof; and

(iii) one or more anti-microbial compound.

The at least three components may be supplied to the mammal as a prepared composition (comprising at least two, preferably three of the (i) one or more sesquiterpene alcohol compound; (ii) one or more polymyxin compound or a derivative thereof; and (iii) one or more anti-microbial compound, as described herein); and/or as a combined composition comprising the at least two, preferably three of the (i) one or more sesquiterpene alcohol compound; (ii) one or more polymyxin compound or a derivative thereof; and (iii) one or more anti-microbial compound, as described herein, as separate components.

A preferred embodiment of the present invention relates to a combinational product comprising : one or more sesquiterpene alcohol compound; one or more polymyxin compound or a derivative thereof; and one or more anti-microbial compound.

In one embodiment of the present invention the antibacterial product provided inhibits proliferation of gram-positive bacteria, such as known pathogenic gram-positive bacteria including but not limited to Staphylococcus spp. (in particular Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus), and/or Streptococcus spp. (in particular Streptococcus pneumoniae, Streptococcus pyogenes and/or Streptococcus agalactiae). In another embodiment of the present invention the antibacterial product provided inhibits proliferation of gram-positive bacteria, such as known pathogenic gram-positive bacteria including but not limited to Enterococcus spp. (in particular Enterococcus faecalis, and/or Enterococcus faecium), Bacillus spp. (in particular Bacillus anthracis, Bacillus cereus), Clostridium spp. (in particular Clostridium botulinum, Clostridium perfringens, Clostridium difficile, Clostridium tetani), Listeria spp. (in particular Listeria monocytogenes), Ureaplasma spp. (in particular Ureaplasma urealyticum) and/or Corynebacterium spp. (in particular Corynebacterium diphtheriae).

In particular the antibacterial product provided inhibits proliferation of gram-positive bacteria selected from one or more of Clostridioides spp. (in particular Clostridioides difficile), Enterococcus spp. (in particular Enterococcus faecalis and/or Enterococcus faecium), Staphylococcus spp. (in particular Staphylococcus aureus), and/or Streptococcus spp. (in particular Streptococcus pneumoniae).

In one embodiment of the present invention the antibacterial product provided inhibits proliferation of gram-negative bacteria, such as known pathogenic gram-negative bacteria including but not limited to Acinetobacter spp. (in particular Acinetobacter baumannii), Bartonella spp. (in particular Bartonella henselae, Bartonella quintana, Bordetella pertussis), Brucella spp. (in particular Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis), Campylobacter spp. (in particular Campylobacter jejuni), Escherichia coli, Enterobacteriaceae spp. Francisella spp. (in particular Francisella tularensis), Haemophilus spp. (in particular Haemophilus influenzae), Helicobacter spp. (in particular Helicobacter pylori), Neisseria spp. (in particular Neisseria gonorrhoeae, Neisseria meningitidis), Chlamydia spp. (in particular Chlamydia trachomatis or Chlamydia psittaci), Pseudomonas spp. (in particular Pseudomonas aeruginosa), Salmonella spp. (in particular Salmonella typhi, Salmonella typhimurium), Shigella spp. (in particular Shigella sonnei), Vibrio spp. (in particular Vibrio cholerae), Yersinia spp. (in particular Yersinia pestis, Yersinia enterocolitica, Yersinia pseudotuberculosis), Borrelia spp. (in particular Borrelia burgdorferi, Borrelia garinii, Borrelia afzelii, Borrelia recurrentis), Legionella spp. (in particular Legionella pneumophila), Leptospira spp. (in particular Leptospira interrogans, Leptospira santarosai, Leptospira weilii, Leptospira noguchii), Rickettsia spp. (in particular Rickettsia rickettsia), Klebsiella spp. (in particular Klebsiella pneumoniae), and/or Treponema spp. (in particular Treponema pallidum).

In yet an embodiment of the present invention the gram-negative bacteria may be selected from the group consisting of Acinetobacter spp. (in particular Acinetobacter baumannii), Bordetella spp. (in particular Bordetella pertussis), Campylobacter spp, Citrobacter spp, Haemophilus spp. (in particular Haemophilus influenzae), Helicobacter spp. (in particular Helicobacter pylori), Klebsiella spp. (in particular Klebsiella pneumoniae), Morganella spp, Mycoplasma spp. (in particular Mycoplasma genitalium), nontuberculous mycobacterium, Neisseria spp. (in particular Neisseria gonorrhoeae), Proteus spp, Pseudomonas spp. (in particular Pseudomonas aeruginosa), and/or the Enterobacterales order (in particular Enterobacter spp, Salmonella spp, Escherichia coli, Salmonella Typhi and/or and/ Salmonella Paratyphi and/or Shigella spp.), Serratia spp.

The mycobacteria may preferably be mycobacterium tuberculosis.

The bacterial infections to be treated by the antibacterial product provided by the present invention can be chosen from any of the infections caused by any of the above-mentioned or other pathogenic bacteria.

One embodiment of the present invention provides an antifungal product. Preferably, the antifungal product according to the present invention may inhibit proliferation of one or more fungal species, preferably a pathogenic fungus species. Furthermore, the antifungal product provided by the present invention can be used for treatment of fungal infections.

In an embodiment of the present invention the fungal species may be selected from the group consisting of Aspergillus spp. (like Aspergillus fumigatus), Blastomyces dermatitidis, Candida spp., (in particular Candida albicans, Candida glabrata, Candida parapsilosis, Candida auris, Candida tropicalis), Cryptococcus spp. (in particular Cryptococcus neoformans, Cryptococcus gattii), Coccidioides spp. (in particular Coccidioides immitis, Coccidioides posadasii), Histoplasma capsulatum, Pneumocystis jirovecii or Stachybotrys spp.

The fungal infections to be treated by the antifungal product provided by the present invention can be chosen from any of the infections caused by any of the above-mentioned or other pathogenic fungus.

The composition according to the present invention may be administered to the mammal intravenously, rectally, topically, nasally, buccally, Sublingually, trans-mucosally, trans- dermally, by inhalation, by injection or intrathecally and/or orally. The injection can be intramuscular, intravenous, intrapulmonary, intramuscular, intradermal, intraperitoneal, intrathecal, or Subcutaneous injection. Preferably, the composition according to the present invention may be administered to the mammal intravenously and/or rectally. More preferably the composition according to the present invention may be administered to the mammal intravenously. The mammal may preferably be a human, a pet or a production animal. Preferably, the pet may be a dog, a cat, or a horse. The production animal may be a ruminant or a pig.

Preferably, the the mammal according to the present invention may be a human or a pet.

In an embodiment of the present invention the composition provided may comprises a carrier material, preferably a pharmaceutical acceptable carrier material.

In one embodiment of the present invention the composition provided comprises a carrier material, wherein the carrier material is suitable for carrying the ingredients selected from the combination of at least two of the following components: one or more sesquiterpene alcohol compound; one or more polymyxin compound or a derivative thereof; and/or one or more nisin-compound.

In an embodiment the composition according to the present invention may be suitable for reducing, limiting and/or prohibiting biofilm formation of microorganism.

The carrier material may preferably be generally safe, non-toxic and neither biologically nor otherwise undesirable. Preferably, the carrier material may be a pharmaceutical acceptable carrier material and may be generally safe, non-toxic and neither biologically nor otherwise undesirable and may be acceptable for veterinary use as well as human pharmaceutical use.

The carrier material may be selected from at least one organic solvent or at least one alcohol; and/or water or an aqueous solution.

In an embodiment of the present invention the aqueous solution comprises suitable and/or pharmaceutical buffers.

The least one organic solvent or at least one alcohol may be DMSO (dimethyl sulfoxide).

The aqueous solution may comprise an acidic aqueous solution, preferably a week acidic aqueous solution.

The pH value of the composition according to the present invention may be controlled by suitable and/or pharmaceutical acceptable buffers. Suitable buffers and/or pharmaceutical buffers according to the present invention may be well known to the skilled person.

The composition according to the present invention comprising the compounds (the one or more sesquiterpene alcohol compound, the one or more polymyxin compound or a derivative thereof and the one or more anti-microbial compound), may be provided as an oral composition, an intravenous composition, a topical composition, or a rectal composition.

In an embodiment of the present invention the carrier material may be selected from solutions that allow the transfer of the drugs intravenously to the patient, such as but not limited to saline water. The different drugs may be dissolved in separate solutions so concentrations can be adapted to the infections.

Preferably, the intravenous composition may be a fluid composition, or an aqueous composition.

The oral composition may be a fluid composition, an aqueous composition, a tablet, a gel, a cream, a lotion, a powder.

The topical composition may be a gel, a cream, a lotion,

The rectal composition may be a fluid composition, an aqueous composition, a tablet, a gel, a cream, a lotion, a suppository

One embodiment of the present invention provides a pharmaceutical agent comprising a composition comprising at least two of the three compounds listed below: one or more sesquiterpene alcohol compound; one or more polymyxin compound or a derivative thereof; and/or one or more nisin-compound.

In an embodiment of the present invention the composition comprises: one or more sesquiterpene alcohol compound; one or more polymyxin compound or a derivative thereof; and one or more anti-microbial compound; and at least one of: a carrier material, and/or a pH-value below 7.0, such as below 6.5, e.g. below 6.0, such as below 5.5, e.g. below 5.0, such as below 4.5, e.g. below 4.0, such as below 3.5, e.g. below 3.0, such as below 2.5.

Another embodiment of the present invention provides a pharmaceutical agent comprising a composition according to any of the above-mentioned embodiments.

A preferred embodiment of the present invention relates to a method for providing a composition according to the present invention, wherein the method comprises the steps of: a. providing one or more sesquiterpene alcohol compound; b. providing one or more polymyxin compound or a derivative thereof; and c. providing one or more anti-microbial compound; d. combining the compounds provided in steps a, b and c - providing the composition according to the present invention.

In an embodiment of the present invention the compounds provided in steps a, b, and c may be individually dissolved.

The individually dissolved compounds may be mixed into a composition according to the present invention or the individually dissolved compounds may be provided individually as a combined composition.

Combining the compounds provided in steps a, b and c - providing the composition according to the present invention may by:

(1) mixing of the compounds provided in steps a, b, and c, providing a single combined composition comprising at least the three compounds provided; or

(2) without mixing the three compounds provided in steps a, b and c, but providing the three compounds provided in steps a, b and c as individually and/or separate compounds.

In an embodiment of the present invention the compounds provided in steps a, b and c may be mixed or combined in any order or sequence. In an embodiment of the present invention the individually dissolved compounds (when not mixed) may be provided to a patient at different time intervals, to a mammal as individually dissolved compounds.

In an embodiment the composition of the present invention may comprise a combination of at least two, preferably three, of one or more sesquiterpene alcohol compound; one or more polymyxin compound or a derivative thereof; and one or more anti-microbial compound; as a mix of the at least two, preferably three, of one or more sesquiterpene alcohol compound; one or more polymyxin compound or a derivative thereof; and one or more anti-microbial compound; or (without mixing) the at least two, preferably three, of one or more sesquiterpene alcohol compound; one or more polymyxin compound or a derivative thereof; and one or more anti-microbial compound are provided individually and/or separately.

Preferably, the composition of the present invention may comprise a combination of at least two, preferably three, of one or more sesquiterpene alcohol compound; one or more polymyxin compound or a derivative thereof; and one or more anti-microbial compound; as a mix of the at least two, preferably three, of one or more sesquiterpene alcohol compound; one or more polymyxin compound or a derivative thereof.

In an embodiment of the present invention the combining of the compounds provided in steps a, b, and c may be mixed into one compound or wherein the compounds kept unmixed as individual and/or separate compounds.

Preferably, the one or more sesquiterpene alcohol compound, such as farnesol, may be provided as a liquid. The one or more sesquiterpene alcohol compound, such as farnesol, may be slightly soluble in water or even insoluble in water.

Solubility of the one or more sesquiterpene alcohol compound, such as farnesol, may be improved by using or adding at least one organic solvent or at least one alcohol. Preferably, the at least one organic solvent or at least one alcohol is miscible with water and a wide range of organic solvents. Preferably, at least one organic solvent or at least one alcohol may be selected from chloroform, dichloromethane, ethyl acetate, DMSO, acetone, ethanol, or the like.

In an embodiment of the present invention the one or more sesquiterpene alcohol compound, such as farnesol, may be solubilised in a liquid comprising a combination of: at least one organic solvent or at least one alcohol; and water or an aqueous solution (the aqueous solution may comprise an acidic aqueous solution).

For improving the solubility, the one or more sesquiterpene alcohol compound, such as farnesol, the temperature of the compound may be increased, preferably the temperature may be increased to a temperature in the range of 20-45°C, such as in the range of 25- 43°C, e.g. in the range of 30-40°C, such as in the range of 34-38°C. e.g. about 37°C.

If shaking is not considered sufficient to provide proper dissolution, the one or more sesquiterpene alcohol compound, such as farnesol, shaking in the ultrasonic bath may be performed.

The one or more polymyxin compound or a derivative thereof may preferably be provided as a salt of the one or more polymyxin compound or a derivative thereof. The one or more polymyxin compound or a derivative thereof may preferably be provided as a powder. Preferably, the one or more polymyxin compound or a derivative thereof may be soluble in water, or in an aqueous solution (the aqueous solution may comprise an acidic aqueous solution) or PMBN may be dissolved in DMSO or a similar solvent or an organic solvent or an alcohol.

The one or more anti-microbial compound, such as a nisin compound, may be provided as a powder. Preferably, one or more anti-microbial compound, such as a nisin compound, may be soluble in water, or in an aqueous solution (the aqueous solution may comprise an acidic aqueous solution). Preferably, the water or the aqueous solution for dissolving the one or more anti-microbial compound, such as a nisin compound, may be provided with an acid, e.g. in dilute acids (such as 0,02 N HCI) or nisin may be dissolved in DMSO.

The dissolved compounds (one or more sesquiterpene alcohol compound; one or more polymyxin compound or a derivative thereof; and one or more anti-microbial compound) may be mixed together after being dissolved providing a miscible composition.

Preferably, the composition comprises a homogeneous or substantially homogenous distribution of the compounds (one or more sesquiterpene alcohol compound; one or more polymyxin compound or a derivative thereof; and one or more anti-microbial compound).

Preferably, the compounds a, b and c may be dissolved in the liquid medium, preferably, the liquid media may be an aqueous solution. The aqueous solution may comprise or the like. Preferably, the liquid medium comprises water, DMSO or a combination hereof.

Preferably, the one or more sesquiterpene alcohol compound, in particular the farnesol compound, provided in step (a), may be provided in a concentration in the composition in the range of 0.001-600 pg/ml, such as in the range of 0.005-450 pg/ml, e.g. in the range of 0.01-300 pg/ml, such as in the range of 0.025-150 pg/ml, e.g. in the range of 0.05-75 pg/ml, such as in the range of 0.075-25 pg/ml, e.g. in the range of 0.1-10 pg/ml, such as in the range of 0.15-5 pg/ml, e.g. in the range of 0.2-2 pg/ml, such as in the range of 0.25-1 pg/ml, e.g. in the range of 0.3-0.75 pg/ml, such as in the range of 0.035-0.5 pg/ml, e.g. in the range of 0.01-0.1 pg/ml, such as in the range of 0.025-0.05 pg/ml

Preferably, the or more polymyxin compound or a derivative thereof, in particular polymyxin B nonapeptide (PMBN), provided in step (b), may be provided in a concentration in the composition in the range of 0.001-600 pg/ml, such as in the range of 0.005-450 pg/ml, e.g. in the range of 0.01-300 pg/ml, such as in the range of 0.025-150 pg/ml, e.g. in the range of 0.05-75 pg/ml, such as in the range of 0.075-25 pg/ml, e.g. in the range of 0.1-10 pg/ml, such as in the range of 0.15-5 pg/ml, e.g. in the range of 0.2-2 pg/ml, such as in the range of 0.25-1 pg/ml, e.g. in the range of 0.3-0.75 pg/ml, such as in the range of 0.035-0.5 pg/ml, e.g. in the range of 0.01-0.1 pg/ml, such as in the range of 0.025- 0.05 pg/ml

Preferably, the one or more anti-microbial compound, e.g. the one or more nisin- compound, in particular nisin variant Z, provided in step (c), may be provided in a concentration in the composition in the range of 0.001-600 pg/ml, such as in the range of 0.005-200 pg/ml, e.g. in the range of 0.01-100 pg/ml, such as in the range of 0.025-50 pg/ml, e.g. in the range of 0.05-75 pg/ml, such as in the range of 0.075-25 pg/ml, e.g. in the range of 0.1-10 pg/ml, such as in the range of 0.15-5 pg/ml, e.g. in the range of 0.2-2 pg/ml, such as in the range of 0.25-1 pg/ml, e.g. in the range of 0.3-0.75 pg/ml, such as in the range of 0.035-0.5 pg/ml, e.g. in the range of 0.01-0.1 pg/ml, such as in the range of 0.025-0.05 pg/ml.

TERMS, ABBREVIATIONS AND DEFINITIONS

List of abbreviations

EDTA: Ethylenediaminetetraacetic acid. OD₆QQ : Optical density 600 - Light is transmitted through a 96 well plate at 600 nm. The amount of light passing through the sample is measured. The amount of light absorbed by the control is subtracted and the remaining absorbance is caused by growth of bacteria or fungi. Higher numbers indicate increased growth. PMBN : Polymyxin B nonapeptide.

MIC: Minimum inhibitory concentration (MIC) is the lowest concentration of a chemical, usually a drug, which prevents visible growth of a bacterium or bacteria. MIC depends on the microorganism, the affected human being (in vivo only), and the antibiotic itself. It is often expressed in micrograms per milliliter (pg/mL) or milligrams per liter (mg/L).

Terms and definitions

Nisin

Nisin is a polycyclic peptide that may be produced by the bacterium Lactococcus lactis. Nisin is known by the skilled person to have antibacterial effect and is widely used as a food preservative. It has 34 amino acid residues, including the uncommon amino acids lanthionine (Lan), methyllanthionine (MeLan), didehydroalanine (Dha), and didehydroaminobutyric acid (Dhb).

Nisin may be found in eight natural variants: A, Z, F, Q, H, J, P, U. Nisin A and Z are similar in terms of inhibition of bacteria. Nisin A and Z are very similar with one difference in that Nisin Z have a higher solubility in water at physiological pH, than Nisin A.

While in general most bacteriocins inhibit only closely related species, nisin is a rare example of a "broad-spectrum" bacteriocin effective against many Gram-positive organisms, including lactic acid bacteria (commonly associated to avoid food spoilage), Listeria monocytogenes (a known pathogen), Staphylococcus aureus, Bacillus cereus, Clostridium botulinum, etc. It is also particularly effective against spores. Gram-negative bacteria are protected by their outer membrane but may become susceptible to nisin action after a heat shock or when nisin is used with a permeabilizer such as the chelator EDTA. When used in combination with EDTA, nisin has the ability to inhibit gram negative bacteria such as E. coli and Salmonella enterica. Nisin, as a class I bacteriocin, is very stable at acidic pHs and is more heat stable at lower pHs. The mode of action of Nisin against pathogens such as L. monocytogenes is to form a pore in the cell wall to dissipate the membrane potential and pH gradient.

There have been studies focusing on the antibacterial effect of different natural variations of Nisin, showing difference in MIC for different variants of Nisin as well as against different bacteria. Furthermore, different mutations of the natural variants of Nisin have been tested, showing that different mutations in the hinge-region (position 20-22) can have great effect on the antibacterial effect of Nisin. Further studies have shown similar effect by mutations in positions 4-6, 12, and/or 29.

PMBN

Polymyxin B is an antibiotic, that may be derived from the bacterium Paenibacillus polymyxa (formerly known as Bacillus polymyxa), that is useful for many Gram-negative infections, while it has not shown useful against Gram positive infections. PMBN can be created by enzymatic modification of polymyxin B.

Polymyxin E, NAB7061, NAB741 or a derivate of these drugs or a derivative of PMBN may be used as substitute for PMBN according to the present invention.

PMBN administered orally or by inhalation, or it can be given by injection into a vein, muscle, or cerebrospinal fluid. Use of PMBN can lead to side effects, such as kidney problems, neurological problems, fever, itchiness, and rash. Injections into muscle may result in significant pain. Other serious side effects may include fungal infections, anaphylaxis, and muscle weakness.

PMBN may have the ability to permeabilize the outer membrane of Gram-negative bacteria. PMBN may alter the bacterial outer membrane permeability by binding to a negatively charged site in the lipopolysaccharide layer, which has an electrostatic attraction for the positively charged amino groups in the cyclic peptide portion (this site normally is a binding site for calcium and magnesium counter ions); the result is a destabilized outer membrane. PMBN may dissolve the fatty acid portion in hydrophobic region of cytoplasmic membrane and disrupts membrane integrity, leading to leakage of cellular molecules and inhibition of cellular respiration. PMBN may further bind and inactivate endotoxin. PMBN may preferably be nonspecific for cell membranes of any type and may therefore considered highly toxic.

PMBN may lack the direct antibacterial activity (i.e. the ability to inhibit bacterial growth), but by its effect on the cell membrane of the bacteria it is able to sensitize (i.e. make sensitive or, as also termed, make susceptible) the bacteria to many antibacterial agents such as hydrophobic antibiotics as well as large antibiotics and some other noxious agents.

It has been shown that a structurally further reduced derivative polymyxin B octapeptide (PMBO) may retain a very effective permeabilizing activity, while polymyxin B heptapeptide (PMBH) may also be active but, less active. Studies have demonstrated that PMBN with five charges may be a lot less toxic than polymyxin B and E with three charges. PMBN, PMEN and PMBO have five (5) positive charges while PMBH has only four (4) positive charges. The attachment of the octanoyl residue to the N - terminus of the residue R4 of the polymyxin B heptapeptide may result in a compound having only three (3) positive charges. Octanoyl polymyxin B heptapeptide may inhibit the growth of bacteria only at a very high concentration (128 ug/ml), whereas the other derivatives such as octanoyl polymyxin B octapeptide and octanoyl polymyxin B nonapeptide, both having four charges (4) were very potent agents to inhibit bacterial growth.

Thus, preferred examples of polymyxin and polymyxin derivatives may include, but are not limited to, Polymyxin B Nonapeptide, Tetra-(Boc)Polymyxin B Nonapeptide, Colistin (Polymyxin E) Nonapeptide, Tetra-(Boc)Colistin (Polymyxin E) Nonapeptide, Tri- (Boc)Polymyxin B Heptapeptide, Penta-(Boc)Polymyxin B Decapeptide, Thr(O-' Bu) Tetra- (N-Boc)Polymyxin B Nonapeptide, Thr (O-'Bu)Penta-(N-Boc)Polymyxin B decapeptide.

Examples of preferred polymyxin B nonapeptides or polymyxin B nonapeptides derivatives and methods of preparation of polymyxin B nonapeptide derivatives may be found in US 2016/222061 Al, which is hereby incorporated by reference in its entirety.

Farnesol

Farnesol is a natural 15-carbon organic compound which is an acyclic sesquiterpene alcohol. Under standard conditions, it is a colorless liquid. Farnesol is a natural pesticide for mites and is a pheromone for several other insects. Other sesquiterpenoides, such as not limited to: nerolidol, bisabolol or apritone; or a combination hereof.

Farnesol may be produced from 5-carbon isoprene compounds in both plants and animals. Phosphate-activated derivatives of farnesol are the building blocks of possibly all acyclic sesquiterpenoids.

It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention.

All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety.

The invention will now be described in further details in the following non-limiting examples. EXAMPLES

General methods

Methods for preparing Nisin, Polymyxin B nonapeptide (PMBN), and

Farnesol, respectively are well known in the art. In the below we highlight examples of known methods to prepare said drug substances.

Nisin is an antimicrobial polypeptide produced by strains of Lactococcus lactis subsp. lactis obtained from e.g., sauerkraut or other fermented product, such as supplemented whey permeate (SWP) medium.

Nisin can be purchased from Sigma Aldrich (St. Louis, MO) for experiments for examples 1-7, but for example 8 and forward the concentrated nisin used in this study were purchased from Handary (Brussel, Belgium).

Polymyxin B nonapeptide (PMBN) was purchased from Sigma Aldrich (St. Louis, MO).

Farnesol - is produced from isoprene compounds in both plants and animals, such as many essential oils such as citronella, neroli, cyclamen, lemon grass, tuberose, rose, musk, balsam, and tolu. It is used in perfumery to emphasize the odours of sweet, floral perfumes and is therefore commercially available.

Farnesol was purchased from Sigma Aldrich (St. Louis, MO).

Assay 1: Measuring drug substance effect on Candida auris (C. auris) growth

Step 1 : Preparation of C. auris for testing

50 g YPD medium (item number: 790130-1 - Bacteriological peptone: 20 g/L - Glucose: 20 g/L - Yeast extract: 10 g/L) from Frederiksen Scientific (Olgod, Denmark) were dissolved in 1 L demineralised water and placed in 100 mL glass bottles. The bottles were auto cleaved at 121°C.

Candida auris DSM 21092 was purchased from DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Liebniz, Germany). The glass vile was opened and the sample placed in YPD medium. After 30 minutes an agar plate were streaked with the medium with C. auris and 1 mL medium with C. auris were added to 100 mL YPD medium and placed at 30°C in a Gallenkamp Plus Oven (Apeldoorn, Holland) at 30°C. overnight with the agar plate. Step 2: Preparation of samples, negative and positive controls

Two test compounds were tested on each plate run, for illustration purposes we call them test compound A (#A) and test compound B (#B) in this Assay.

5 mg of the test compounds were weighed at an analytical balance AND FR-300 and YPD medium were added to a drug concentration of 1000 pg/mL, and both drugs were serially diluted in YPD medium, with #A added at 50 pl to each well starting at a concentration of 0 pg/mL at row A and then increasing concentration towards row H in the horizontal direction. #B was added at 50 pl to each well starting with an initial concentration of 0 pg/mL at column 1 and then increasing concentrations towards column 7.

For C. auris cultures OD₆oo of 0.5 equals 10⁷cells/ml. so, the media with fungi were diluted to OD₆QO = 0.5, and then subsequently diluted 1 : 100 with YPD medium to a final concentration of 10⁵ cfu/ml. 50 pL of the stock solution were added to each well in the 96 well plate.

150 pl of YPD medium with C. auris were added to four wells for a positive control. The negative control were four wells with 150 pl of YPD medium with 10 pg/mL Posaconazole from Sigma Aldrich (St. Louis, MO) (Item number: 32103-25MG).

Depending on the tested compound the concentrations tested varied depending on the test compound's effects on the experimental setup and method for detection.

The following table illustrates one experimental setup of a plate tested in a series of 3.

Table 1 - Concentrations of tested test compound expressed in the concentration of test compound added in pg/mL of each test compound, indicated in each cell symbolizing a well also comprising C. auris as described in this assay. In this illustrative plate setup, the test compound A (#A) was PMBN, and test compound B (#B) was Farnesol.

Step 3: Testing the effect on C. auris growth of the test compounds (24hrs incubation') After addition of the test compounds diluted by YPD media and controls consisting of YPD media, the fungi were allowed to incubate for 24 hours at 30°C.

The OD₆QO were measured at 600 nm for the plate in a VersaMax tunable Microplate reader from Molecular Devices Corporation (San Jose, CA) using the software SoftMax Pro also from Molecular Devices Corporation. The plate was shaken by the reader before reading.

Step 4: Calculation and normalization of the QD600 readings

The readings for position Al should correspond to the average for the positive control and this were checked. From all the readings were subtracted the average of the negative control, which correspond to OD₆oo for the medium, leaving OD₆oo for the bacteria.

All data were normalized by dividing all numbers by the OD₆oo for position Al and measured in %. Inhibition is reduction below 100% in Al for any of the wells and a low number correspond to an effective inhibition by the drug or drugs involved.

The detailed setup of experiments, including sample concentrations and specific drug substances used, alone or in combination are described in the following examples, where it is also explained which assay setup is utilized.

Assay 2: Measuring Drug Substance effect on Escherichia coli (E. coli) growth

Step 1 : Preparation of E. co// for testing

20 g LB medium from Sigma Aldrich (St. Louis, MO) were dissolved in 1 L demineralised water and placed in 100 mL glass bottles. The bottles were auto cleaved at 121°C.

E. coli DSM 15174 was purchased from DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Liebniz, Germany). The glass vile was opened and the sample placed in LB medium. After 30 minutes a LB agar plate was streaked with the medium with E. coli and 1 mL medium with E. coli were added to 100 mL LB medium (Lennox) (NaCI: 5 g/L - Tryptone: 10 g/L - Yeast Extract: 5 g/L) and placed at 37°C in a in a Memmert incubator (Berlin, Germany) over night with the agar plate.

Step 2: Preparation of samples, negative and positive controls

Two test compounds were tested on each plate run, for illustration purposes we call them test compound A (#A) and test compound B (#B) in this Assay. 5 mg of the test compounds were weighed at an analytical balance AND FR-300 and LB medium were added to a drug concentration of 1000 pg/mL, and both drugs were serially diluted in LB medium, with #A added at 50 pl to each well starting at a concentration of 0 pg/mL at row A and then increasing concentration towards row H in the horizontal direction. #B was added at 50 pl to each well starting with an initial concentration of 0 pg/mL at column 1 and then increasing concentrations towards column 8.

For E. coli cell cultures, OD₆oo of 1.0 equals 8 x 10⁸cells/ml, so the media that had grown over night with bacteria were diluted to OD₆oo = 1, and then subsequently diluted to a final concentration of 10⁵ cfu/ml. 50 pL of the of media with E. coli was added to each well with dissolved drugs in the 96 well plate.

The negative control was four wells with 150 pl of LB medium with 100 pg/mL chloramphenicol from Sigma Aldrich (St. Louis, MO) (Item number: C0378-25G) in column 11 at the four upper rows.

150 pl LB medium with E. coli were added to 4 wells for positive control at column 12 at the upper four rows.

Depending on the tested compound the concentrations tested vary depending on the test compound's effects on the experimental setup and method for detection.

Table 2 - Concentrations of tested test compound expressed in the concentration of test compound added in pg/mL of each test compound, indicated in each cell symbolizing a well also comprising E. coli as described in this assay.

In this illustrative plate setup, the test compound A (#A) was PMBN, and test compound B (#B) was Farnesol.

Step 3: Testing the effect on E. coli growth of the test compounds (24hrs incubation') After addition of the test compounds dissolved in LB media and controls in LB media, the bacteria were allowed to incubate for 24 hours at 37°C.

The OD600 were measured at 600 nm for the plate in a VersaMax tunable Microplate reader from Molecular Devices Corporation (San Jose, CA) using the software SoftMax Pro also from Molecular Devices Corporation. The plate was shaken by the reader before reading.

Step 4: Calculation and normalization of the QD600 readings

All data were normalized by dividing all numbers by the OD₆oo for position Al and measured in %.

Inhibition is reduction below the 100% in Al for any of the wells and a low number correspond to an effective inhibition by the drug or drugs involved.

The detailed setup of experiments and sample concentrations and specific drug substances used, alone or in combination are described in the examples.

Example 1: Effect of Nisin and Polymyxin B nonapeptide (PMBN) alone or in combination on the growth of Candida auris DSM 21092 (C. auris)

To evaluate the effect of Nisin from Sigma Aldrich (St. Louis, MO) (Item number: N5764- 1G) and/or PMBN from Sigma Aldrich (St. Louis, MO) (Item number: P2076-5MG) on C. auris growth, samples and controls of this experiment were set up and evaluated according to Assay 1, where a series of Nisin and PMPN concentrations were tested. 5 mg. of each drug were measured at an analytical AND FR-300 balance and diluted with YPD medium to 1000 pg/mL in a 15 mL falcon tube. Nisin stock solution at 1000 pg/mL in YPD medium were diluted by YPD medium to concentrations: 0, 3, 6, 12, 25, 50 and 100 |jg/mL, which were applied as previously described with Nisin added by columns.

PMBN stock solution at 1000 pg/mL in YPD medium were diluted by YPD medium to concentrations: 0, 1, 4, 16, 63, 250 and 1000 pg/mL, which were applied as previously described with PMBN added by rows.

Each drug was added with 50 pL to each well with 50 pL of media with C. auris for a final concentration at 1/3 of the concentrations mentioned above.

The tested concentrations are indicated, below in table 3, which also shows the results determined according to Assay 1 and based on 3 repetitions of the experimental setup. In table 3 Nisin is abbreviated as N and PMBN as P.

Table 3. Effect on C. auris growth of Nisin alone or in combination with PMBN at different concentrations, alone or in combination. Each cell represents the relative growth in %. The %-growth is normalized relative to the C. auris growth and OD600 read at 0.0 pg/mL Nisin and 0.0 pg/mL PMPN. Positive control: 91%, Negative control: 15%

Results/conclusions:

The results shown in the above table show that Nisin alone was not efficient in inhibiting C. auris growth in the interval between 1.0 and 16.7 (pg/mL) that confirms what literature has previously reported, that the Nisin concentration in comparable setups needs to be at least 68-300 pg/mL to show significant effect. There is however some effect of Nisin alone in the highest concentration of 33.3 (pg/mL). To our knowledge, PMBN has not been tested in similar setups against C. auris or any other Candida fungi or any other fungi, however, from Table 3 it is evident that PMBN was not an efficient stand-alone inhibitor of C. auris growth as shown in the above table, at least not in lower concentrations. However, when added in 333 pg/mL, there was an inhibiting effect.

The surprising effect is the synergy between the two compounds used. As demonstrated, even when low concentration of PMBN was added to the highest concentration of Nisin there was evidence of significant inhibition of C. auris. Furthermore, the result demonstrates a significant drop in inhibition when only 1 pg/mL Nisin was added to any PMBN concentration. This indicates a surprising synergy between the two drugs. The best results were achieved when combining the highest tested concentrations of both drug substances achieving a very high reduction of growth which was not expected when the experiments were set up and clearly demonstrating the surprising synergic effect of the combination of the two substances.

Example 2: Effect of Nisin and Farnesol alone or in combination on the growth of Candida auris DSM 21092 (C. auris}

To evaluate the effect of Nisin and/or Farnesol on C. auris growth, samples and controls of this experiment were set up and evaluated according to Assay 1, where a series of Nisin and Farnesol concentrations were tested.

Nisin from Sigma Aldrich (St. Louis, MO) stock solution at 1000 pg/mL in YPD medium were diluted by YPD medium to concentrations: 0, 3, 6, 12, 25, 50 and 100 pg/mL, which were applied as previously described with Nisin added by columns.

Farnesol from Sigma Aldrich (St. Louis, MO) stock solution at 1000 pg/mL in YPD medium were diluted by YPD medium to concentrations: 0, 1, 4, 16, 63, 250 and 1000 pg/mL, which were applied as previously described with Farnesol added by rows.

The tested concentrations are indicated in table 4, below, which also shows the results determined according to Assay 1 and based on 3 repetitions of the experimental setup. Farnesol is abbreviated F and Nisin is abbreviated N in table 4 below.

Table 4: Effect on C. auris growth of Nisin alone or in combination with Farnesol at different concentrations, alone or in combination. Each cell represents the relative growth in %. The %-growth is normalized relative to the C. auris growth and OD600 read at 0.0 pg/mL Nisin and 0.0 pg/mL Farnesol. Positive control: 88%, Negative control: 8%

Results/conclusions:

The results shown in table 4 show that Nisin alone was not efficient in inhibiting C. auris growth in the used concentration interval and thus confirms what literature has previously reported, that the Nisin concentration in comparable setups needs to be at least 68-300 pg/mL to show effect.

When testing different concentrations of Farnesol, in this assay, it was evident that it would only effectively inhibiting C. auris growth at high concentrations, i.e., above 83 pg/mL, though some effect can be seen at lower concentration. Literature reports Farnesol as inhibiting C. auris in concentrations above 20 pg/mL.

However, when the two drug substances were combined a surprising synergistic effect was evident and pronounced. The addition of low concentrations of Nisin, which did not inhibit the growth of C. auris to a meaningful extent on its own, to low concentrations of Farnesol, which also did not affect the C. auris growth independently, clearly enabled an inhibition of the growth to below 70% in most concentration combinations. The best results were achieved when combining the highest tested concentrations of both drug substances achieving a very high reduction of growth which was not expected when the experiments were set up and clearly demonstrating the surprising synergic effect of the combination of the two substances. Example 3: Effect of Polymyxin B nonapeptide (PMBN) and Farnesol alone or in combination on the growth of Candida auris DSM 21092 (C. auris)

To evaluate the effect of Farnesol and PMBN on C. auris growth, samples and controls of this experiment were set up and evaluated according to Assay 1, where a series of Farnesol and PMBN concentrations were tested according to said assay.

Farnesol from Sigma Aldrich (St. Louis, MO) stock solution at 1000 pg/mL in YPD medium were diluted by YPD medium to concentrations: 0, 1, 4, 16, 63, 250 and 1000 pg/mL, which were applied as previously described with Farnesol added by columns.

PMBN from Sigma Aldrich (St. Louis, MO) stock solution at 1000 pg/mL in YPD medium were diluted by YPD medium to concentrations: 0, 1, 4, 16, 63, 250 and 1000 pg/mL, which were applied as previously described with PMBN added by rows.

The tested concentrations are indicated in table 5 below, which also shows the results determined according to Assay 1 and based on 3 repetitions of the experimental setup. Again, Farnesol is abbreviated as F in table 5, while PMBN is abbreviated as P.

Table 5: Effect on C. auris growth of Farnesol alone or in combination with PMBN at different concentrations, alone or in combination. Each cell represents the relative growth in %. The %-growth is normalized relative to the C. auris growth and OD600 read at 0.0 pg/mL Farnesol and 0.0 pg/mL PMBN. Positive control: 77%, Negative control: 7%

Results/conclusions:

When testing different concentrations of Farnesol, in this assay, it was evident that it would only inhibit C. auris growth at high concentrations, i.e., above 21 pg/mL. This is consistent with literature, which reports Farnesol as having inhibiting properties on C. auris in concentrations above 20 pg/mL.

To our knowledge, PMBN has not been tested in similar setups against C. auris or any other Candida fungi, however, from table 5 it is evident that PMBN is not an efficient stand-alone inhibitor of C. auris growth as shown in the above table. When added in 333 pg/mL, there is an inhibiting effect.

However, when the two drug substances were combined a synergistic effect was evident and pronounced, as demonstrated in table 5. When both Farnesol and PMBN were added in concentrations at 0.3 pg/mL, the inhibition is higher than when PMBN was added in 83.3 pg/mL, and the inhibition is higher than when Farnesol was added alone in 20.8 pg/mL. This clearly is synergistic effect between the two substances and is highly surprising, as previous studies have demonstrated that when farnesol was applied to C. auris farnesol decreased the thickness of C. auris. Other previous studies have also demonstrated that PMBN sensitizes gram negative bacteria to antibiotics that have little or no effect on gram negative bacteria. It has been hypothesized that this was because PMBN altered the physical structure of the outer membrane. Both Farnesol and PMBN may inhibit infections by altering the membrane, and this would indicate that by adding both drugs at the same time an additive effect might be achieved, but it was highly surprising that a strong synergistic effect were achieved. The strong synergistic effect is an additional advantage because it allows the reduction of the use of PMBN which may be somewhat toxic, though much less toxic than polymyxin B.

Example 4: Combined effect of Polymyxin B nonapeptide (PMBN), Farnesol, and Nisin on the growth of Candida auris DSM 21092 (C. auris)

Table 5, see above, demonstrates effective and synergistic inhibition of PMBN and Farnesol on C. auris, while table 4, demonstrates synergistic inhibition of Nisin and Farnesol on C. auris. To evaluate the effect of Nisin on different relevant concentrations of PMBN and Farnesol on C. auris growth, this experiment was set up.

Setup of experiment

The following series of Farnesol and PMBN concentrations were tested and these concentrations were the concentrations tested in the plate: I: P: 20.8 pg/mL./F: 1.3 pg/mL II: P: 5.2 pg/mL./F: 5.2 pg/mL III: P: 1.3 pg/mL./F: 80.8 pg/mL And then a series of different Nisin concentrations were added, where Nisin from Sigma Aldrich (St. Louis, MO) stock solution at 1000 pg/mL in YPD medium were diluted by YPD medium to concentrations: 0, 3, 6, 12, 25, and 50 pg/mL, which were applied as previously described with Nisin added by rows.

Table 6, below, shows the effect of different concentration of Nisin on the growth of C. auris, when added to different concentrations of PMBN and Farnesol.

Table 6: Effect on C. auris growth of different selected concentrations of Farnesol in combination with PMBN at different concentrations, alone or in combination with different concentrations of Nisin. I: PMBN: 20.8 pg/mL and Farnesol: 1.3 pg/mL. II: PMBN : 5.2 pg/mL and Farnesol: 5.2 pg/mL. Ill: PMBN : 1.3 pg/mL and Farnesol: 20.8 pg/mL. Each cell represents the relative growth in %. The %-growth is normalized relative to the C. auris growth and OD600 read at the selected Farnesol and PMBN concentrations and 0.0 pg/mL Nisin. Positive control: 85%, Negative control: 6%

Results/conclusions:

Three different combinations of Farnesol and PMBN were selected from table 5 based on level of inhibition and different concentrations of Farnesol and PMBN. The concentrations of both drugs ranged from 1.3 - 20.8 pg/mL. When Nisin were added to all concentrations it was apparent that just 1 pg/mL added to a mixture of both Farnesol and PMBN inhibited C. auris more than 33-45%, which was significantly more than when 1 pg/mL Nisin was added alone. This clearly demonstrates that there was a surprising synergy between the tree substances when applied together against C. auris, as the inhibition was higher than would be expected from their individual effects added. Example 5: Effect of Posaconazole on the growth of Candida auris DSM 21092 (C. auris)

To evaluate the effect of know antifungal like Posaconazole on C. auris growth as a reference for the effect of Nisin, Farnesol and PMBN, samples and controls of this experiment were set up and evaluated according to Assay 1 with one row, where a series of Posaconazole concentrations were tested.

Posaconazole from Sigma Aldrich (St. Louis, MO) stock solution at 100 pg/mL in YPD medium were diluted by YPD medium to concentrations: 0, 3, 6, 12, 25 and 50 pg/mL, which were applied as previously described with Posaconazole added by columns.

The tested concentrations are indicated table 7 below, which demonstrates the results determined according to Assay 1 and based on 2 repetitions of the experimental setup. Posaconazole is abbreviated as Posa. in table 7 below.

Table 7: Effect on C. auris growth of Posaconazole alone. Each cell represents the relative growth in %. The %-growth is normalized relative to the C. auris growth and OD600 read at 0.0 pg/mL Posaconazole. Positive control: 86%, Negative control: 6%.

Results/conclusions:

From table 7 its apparent that even 1 pg/mL Posaconazole inhibited C. auris significantly and reduced bacterial growth by 80%, and thus, confirms what literature has previously reported, that the Posaconazole concentration at 0.015 - 1.1 pg/mL in comparable setups inhibits various Candida auris strains.

Comparing the results in table 5, 6 and 7 it is apparent that a combination of 83.3 pg/mL Farnesol, 0.3 pg/mL PMBN and 1.0 pg/mL Nisin should inhibit C. auris as well as a designated antifungal like Posaconazole.

Example 6: Effect of Citric acid alone on the growth of Candida auris DSM 21092 (C. auris)

To evaluate the effect of Citric acid from Sigma Aldrich (St. Louis, MO) (Item number: C0759-100G) on C. auris growth as a reference for the effect of Nisin, Farnesol and PMBN. Samples and controls of this experiment were set up and evaluated according to Assay 1 with one row, where a series of Citric acid concentrations were tested according to assay 1. Citric acid from Sigma Aldrich (St. Louis, MO) stock solution at 100 pg/mL in YPD medium were diluted by YPD medium to concentrations: 0, 3, 6, 12, 25, 50 and 100 pg/mL, which were applied as previously described with Citric acid added by columns.

The tested concentrations are indicated in table 8 below, which also shows the results determined according to Assay 1 and based on 2 repetitions of the experimental setup.

Table 8: Effect on C. auris growth of Citric acid alone. Each cell represents the relative growth in %. The %-growth was normalized relative to the C. auris growth and OD600 read at 0.0 pg/mL Citric acid. Positive control: 94%, Negative control 6%

Results/conclusions:

Table 8 demonstrated that Citric acid, which have previously been shown to be an effective antibacterial drug, have only limited inhibiting effect against the fungus C. auris. This is consistent with literature which states that Citric Acid must be applied in concentrations at 30 mg/mL to inhibit Candida.

Example 7: Effect of Polymyxin B nonapeptide (PMBN), Farnesol, and Nisin on the growth of Escherichia coli DSM 15174 (E. co//)

The effect of the combination of Polymyxin B nonapeptide (PMBN), Farnesol, and Nisin on the growth of Escherichia coli (E. coli) was evaluated at different concentrations of Nisin, PMBN and Farnesol.

An initial experiment with different concentrations of each of Polymyxin B nonapeptide (PMBN) (P) - PMBN was obtained from Sigma Aldrich (St. Louis, MO) stock solution at 1000 pg/mL in LB medium, Farnesol (F) - Farnesol was obtained from Sigma Aldrich (St. Louis, MO) stock solution at 1000 pg/mL in LB medium, and Nisin (N) - Nisin was obtained from Sigma Aldrich (St. Louis, MO) stock solution at 1000 pg/mL in LB medium, was performed :

Table 9, below, shows the effect of each of Farnesol (F), PMBN (P) and Nisin (N) on the growth of E. coli, when added individually at different concentrations.

Table 9: Effect on E. coli growth of different concentrations Farnesol, PMBN or Nisin. Each cell represents the relative growth in %. The %-growth was normalized relative to the E. coli growth and OD600 read at the selected Farnesol and PMBN concentrations and 0.0 pg/mL Nisin. Positive control: 73%, Negative control: 6%.

Two concentrations of Farnesol (F) and PMBN (P) were prepared as test composition I or test composition II:

I: P: 20.8 pg/mL./F: 1.3 pg/mL

II: P: 5.2 pg/mL./F: 5.2 pg/mL

Then a series of Nisin concentrations were added to either test composition I or test composition II and tested in the above explained assay.

Nisin from Sigma Aldrich (St. Louis, MO) stock solution at 1000 pg/mL in LB medium were diluted by LB medium to concentrations: 0, 1, 4, 16, 63, and 250 pg/mL, which were applied as previously described with Nisin added by rows.

Table 10, below, shows the effect of different concentration of Nisin on the growth of E. coli, when added to two different concentrations of PMBN and Farnesol (test composition I and test composition II).

Table 10: Effect on E. coli growth of two different concentrations of Farnesol and PMBN in combination with different concentrations of Nisin. I: PMBN : 20.8 pg/mL and Farnesol: 1.3 |jg/mL. II: PMBN : 5.2 pg/mL and Farnesol: 5.2 pg/mL. Each cell represents the relative growth in %. The %-growth was normalized relative to the E. coli growth and OD600 read at the selected Farnesol and PMBN concentrations and 0.0 pg/mL Nisin. Positive control: 73%, Negative control: 6%.

Results/conclusions:

The combination of Farnesol and PMBN and Nisin showed a surprisingly strong inhibition of E. coli - an effect that was much stronger than provided by inhibition provided by each of the ingredients individually. This demonstrated that there was synergistic effect from adding all three drugs together and applying them against E. coli. When 0.3 pg/mL Nisin is added to 20.8 pg/mL PMBN and 1.3 pg/mL Farnesol, Nisin inhibits 60% of the growth, but when nisin is added alone the same amount of Nisin inhibits 10%. When 83.3 pg/mL Nisin is added to 20.8 pg/mL PMBN and 1.3 pg/mL Farnesol Nisin inhibits 94% of the growth added to the inhibition of PMBN and Farnesol, which is 25% normalised in the top line. This synergy was unexpected.

Example 8: Determining the effect of Polymyxin B nonapeptide (PMBN) and Farnesol, on the effect of Nisin on bacterial growth (determined on Enterococcus faecium DSM 25389 and Staphylococcus aureus DSM 113533).

A series of various tests was constructed for demonstrating that the one or more sesquiterpene alcohol compound, in particular Farnesol; and the one or more polymyxin compound or a derivative thereof, in particular PMBN, does not interfere with the action and the effectivity of the one or more anti-microbial compound, in particular Nisin.

Farnesol and polymyxin compound (PMBN) was used in equal amounts in each well ranging in total form 0 to 83.3 pg/ml, and nisin was added in concentrations ranging from 0 to 333.3 pg/ml according to the below tables 10 (Enterococcus faecium) and table 11 (Staphylococcus aureus).

Table 10, shows the effect of Farnesol (F) and PMBN (P) on the effect of Nisin (N) on the growth of Enterococcus faecium, when used at different concentrations.

Table 11, shows the effect of Farnesol (F) and PMBN (P) on the effect of Nisin (N) on the growth of Staphylococcus aureus, when used at different concentrations.

The experiments demonstrates that the one or more sesquiterpene alcohol compound, in particular Farnesol; and the one or more polymyxin compound or a derivative thereof, in particular PMBN, does not interfere, inhibit or limit the effect of the one or more antimicrobial compound, in particular Nisin with a known effect against all gram positive bacteria, when tested at the two gram-positive bacteria, Enterococcus faecium and Staphylococcus aureus.

Example 9: Combined effect of Polymyxin B nonapeptide (PMBN), Farnesol, and Nisin on the growth of various bacteria and fungus.

A series of various tests was constructed for demonstrating the use of the composition according to the present invention comprising the combination of one or more sesquiterpene alcohol compound, in particular Farnesol; one or more polymyxin compound or a derivative thereof, in particular PMBN; and/or one or more anti-microbial compound, in particular Nisin. Farnesol from Sigma Aldrich (St. Louis, MO) stock solution at 1000 pg/mL in YPD medium for fungi and LB medium for bacteria were diluted by relevant medium (e.g. YPD medium) to concentrations between 5 and 1000 pg/mL before dilution in the experiment, as indicated in the table below.

PMBN from Sigma Aldrich (St. Louis, MO) stock solution at 1000 pg/mL in YPD medium for fungi and LB medium for bacteria were diluted by relevant medium (e.g. YPD medium) to concentrations between 5 and 1000 pg/mL before dilution in the experiment, as indicated in the table 12 below.

Nisin from Handary (Brussel, Belgium) (Itim number: 0304 - 10G) stock solution at 1000 pg/mL in YPD medium for fungi and LB medium for bacteria were diluted by relevant medium (e.g. YPD medium) to a concentration of 30 pg/ml or 120 pg/mL before dilution in the experiment, which were applied to the samples, as indicated in the table 12 below.

The composition comprising different concentrations of farnesol, PMBN and Nisin was tested for the effect in inhibiting growth of the following microorganisms; Acinetobacter spp.DSM 586; E.coli DH5a; E. coli DSM 15174; Salmonella enterica subsp. Enterica DSM 11320; and Cryptococcus neoformans DSM 11959.

The synergistic effect of the composition according to the present invention was investigated by determining the Fractional Inhibitory Concentration (FIC) index value.

This Fractional Inhibitory Concentration (FIC) index value is used to determine the impact on potency of the combination of ingredients in a composition relative to their individual activities, based on MIC values.

To quantify the interactions between the ingredients of the composition, the FIC index may be determined according to Eliopoulos, G. and Moellering Jr., R.C. (1996) Antimicrobial Combinations. In: Lorian, V., Ed., Antibiotics in Laboratory Medicine, the Williams 8i Wilkins Co., Baltimore, 330-396. Synergy may be defined as an FIC index of less than 0.5, and antagonism at > 4. The following equation is used :

(A/MICA) + (B/MICB) + (C/MICc) = FICA + FIC_B + FIC_c = FIC index

A = Farnesol concentration

B = PMBN concentration

C = Nisin concentration Synergy may be observed from the FIC index when the combination of compounds results in an FIC index value of <0.5, then the combination of the compounds increases the inhibitory activity (decrease in MIC) of one or more compounds than the compounds alone.

Additive or indifference may be observed from the FIC index when the combination of compounds results in an FIC value of 0.5 - 4, the combination has no increase in inhibitory activity or a slight increase in inhibitory activity from the additive effect of both compounds combined.

Antagonism may be observed from the FIC index when the combination of compounds results in an FIC value of >4, the combination of compounds increases the MIC, or lowers the activity of the compounds.

Results

Table 12: The effect of the composition of the invention against a wide range of microorganisms with synergistic effects.

The results shown in the above table demonstrate that the composition according to the present invention comprising the combination of (i) one or more sesquiterpene alcohol compound, in particular Farnesol; (ii) one or more polymyxin compound or a derivative thereof, in particular PMBN; and (iii) one or more anti-microbial compound, in particular Nisin, has a strong inhibiting effect on a broad range of different microorganisms, including bacteria and fungus.

Example 9 demonstrates that: the combination of (i) one or more sesquiterpene alcohol compound, in particular Farnesol; (ii) one or more polymyxin compound or a derivative thereof, in particular PMBN; and (iii) one or more anti-microbial compound, in particular Nisin demonstrate a synergistic effect against a broad range of microorganisms; the individual concentrations of one or more sesquiterpene alcohol compound, in particular Farnesol; one or more polymyxin compound or a derivative thereof, in particular PMBN may be reduced thus avoiding or reducing the negative effects of high concentrations of the individual compounds; and that the composition according to the present invention comprising the combination of (i) one or more sesquiterpene alcohol compound, in particular Farnesol; (ii) one or more polymyxin compound or a derivative thereof, in particular PMBN; and (iii) one or more anti-microbial compound, in particular Nisin, may be a highly suitable drug for treating unknown microbial infections, with the limited, reduced or no side effects.

The synergy discovered between Nisin, PMBN and farnesol were surprising and enables the inhibition of bacteria with a lower overall concentration of drugs and therefore toxicity than if no synergy existed. Prior to the experiments it could not be predicted that synergy with FICI < 0.5 existed between nisin, PMBN and farnesol, since the molecules acted in an unexpected way in excess of what was predictable.

Claims

1. A composition comprising the combination of at least the following three components:

(i) one or more sesquiterpene alcohol compound;

(ii) one or more polymyxin compound or a derivative thereof; and

(iii) one or more anti-microbial compound.

2. The composition according to the present invention, wherein the composition is a mix of the three components into a single composition or a combination of the three compounds as individual or separate compounds.

3. The composition according to anyone of claims 1 or 2, wherein the anti-microbial compound is a nisin-compound or a derivative thereof.

4. The composition according to anyone of claims 1-3, wherein the composition comprises one or more sesquiterpene alcohol compound and one or more polymyxin compound.

5. The composition according to anyone of the preceding claims, wherein the one or more sesquiterpene alcohol compound is selected from a farnesol compound, a nerolidol compound, an apritone compound, or a combination hereof.

6. The composition according to anyone of the preceding claims, wherein the one or more polymyxin compound is a polymyxin B compound, a polymyxin E compound, or a combination hereof.

7. The composition according to claim 6, wherein the polymyxin B compound is polymyxin B nonapeptide (PMBN).

8. The composition according to anyone of claims 2-7, wherein the one or more nisin compound is a nisin variant A, a nisin variant Z, a nisin variant F, a nisin variant Q, a nisin variant H, a nisin variant J, a nisin variant P, a nisin variant U, a derivative constructed by substituting one or more amino acids, a peptide with a similar molecular structure or a combination hereof.

9. The composition according to anyone of claims 1-8, wherein the composition comprises a content of: one or more sesquiterpene alcohol compound, in particular the farnesol compound, in the composition in the range of 0.001-600 pg/ml, such as in the range of 0.025- 150 pg/ml, e.g. in the range of 0.05-75 pg/ml, such as in the range of 0.075-25 pg/ml, e.g. in the range of 0.1-10 pg/ml, such as in the range of 0.15-5 pg/ml, e.g. in the range of 0.2-2 pg/ml, such as in the range of 0.25-1 pg/ml, e.g. in the range of 0.3-0.75 pg/ml, such as in the range of 0.035-0.5 pg/ml, e.g. in the range of 0.01-0.1 pg/ml, such as in the range of 0.025-0.05 pg/ml; one or more polymyxin compound or a derivative thereof, in particular polymyxin B nonapeptide (PMBN), in the composition in the range of 0.001-600 pg/ml, such as in the range of 0.025-150 pg/ml, e.g. in the range of 0.05-75 pg/ml, such as in the range of 0.075-25 pg/ml, e.g. in the range of 0.1-10 pg/ml, such as in the range of 0.15-5 pg/ml, e.g. in the range of 0.2-2 pg/ml, such as in the range of 0.25-1 pg/ml, e.g. in the range of 0.3-0.75 pg/ml, such as in the range of 0.035-0.5 pg/ml, e.g. in the range of 0.01-0.1 pg/ml, such as in the range of 0.025-0.05 pg/ml; and/or one or more nisin-compound, in particular nisin variant Z, in the composition in the range of 0.001-600 pg/ml, such as in the range of 0.005-200 pg/ml, e.g. in the range of 0.01-100 pg/ml, such as in the range of 0.025-50 pg/ml, e.g. in the range of 0.05-75 pg/ml, such as in the range of 0.075-25 pg/ml, e.g. in the range of 0.1- 10 pg/ml, such as in the range of 0.15-5 pg/ml, e.g. in the range of 0.2-2 pg/ml, such as in the range of 0.25-1 pg/ml, e.g. in the range of 0.3-0.75 pg/ml, such as in the range of 0.035-0.5 pg/ml, e.g. in the range of 0.01-0.1 pg/ml, such as in the range of 0.025-0.05 pg/ml

10. An antibacterial and/or antifungal product comprising the composition according to anyone of claims 1-9.

11. A pharmaceutical agent comprising the composition according to anyone of claims 1-9, for use as a medicament.

12. A pharmaceutical agent comprising the composition according to anyone of claims 1-9, for use in the treatment of a bacterial and/or a fungal infection in a mammal.

13. A pharmaceutical agent comprising the composition according to anyone of claims 1-9, for use in the treatment of bacterial, fungal and/or viral infections such as systemic, cutaneous or pulmonary infections in a mammal.

14. A combinational product comprising: one or more sesquiterpene alcohol compound; one or more polymyxin compound or a derivative thereof; and one or more anti-microbial compound.

15. A method for providing a composition according to claims 1-19, an agent of product according to anyone of claims 10-14, wherein the method comprises the steps of: a. providing one or more sesquiterpene alcohol compound; b. providing one or more polymyxin compound or a derivative thereof; and c. providing one or more anti-microbial compound; d. combining the compounds provided in steps a, b and c.

16. The method according to claim 15, wherein the combining of the compounds provided in steps a, b, and c are mixed into one compound or wherein the compounds kept unmixed as individual and/or separate compounds.

17. The method according to anyone of claims 15-16, wherein the compounds provided in steps a, b, and c are individually dissolved before they are mixed into one compound.

18. The method according to anyone of claims 15-17, wherein the liquid media is an aqueous solution, in particular a DMSO medium.

19. The method according to anyone of claims 15-18, wherein the: one or more sesquiterpene alcohol compound, in particular the farnesol compound, is provided in a concentration in the composition in the range of 0.001-600 pg/ml, such as in the range of 0.025-150 pg/ml, e.g. in the range of 0.05-75 pg/ml, such as in the range of 0.075-25 pg/ml, e.g. in the range of 0.1-10 pg/ml, such as in the range of 0.15-5 pg/ml, e.g. in the range of 0.2-2 pg/ml, such as in the range of 0.25-1 pg/ml, e.g. in the range of 0.3-0.75 pg/ml, such as in the range of 0.035- 0.5 pg/ml, e.g. in the range of 0.01-0.1 pg/ml, such as in the range of 0.025-0.05 pg/ml; one or more polymyxin compound or a derivative thereof, in particular polymyxin B nonapeptide (PMBN), is provided in a concentration in the composition in the range of 0.001-600 pg/ml, such as in the range of 0.025-150 pg/ml, e.g. in the range of 0.05-75 pg/ml, such as in the range of 0.075-25 pg/ml, e.g. in the range of 0.1-10 pg/ml, such as in the range of 0.15-5 pg/ml, e.g. in the range of 0.2-2 pg/ml, such as in the range of 0.25-1 pg/ml, e.g. in the range of 0.3-0.75 pg/ml, such as in the range of 0.035-0.5 pg/ml, e.g. in the range of 0.01-0.1 pg/ml, such as in the range of 0.025-0.05 pg/ml; and/or one or more nisin-compound, in particular nisin variant Z, is provided in a concentration in the composition in the range of of 0.001-600 pg/ml, such as in the range of 0.005-200 pg/ml, e.g. in the range of 0.01-100 pg/ml, such as in the range of 0.025-50 pg/ml, e.g. in the range of 0.05-75 pg/ml, such as in the range of 0.075-25 pg/ml, e.g. in the range of 0.1-10 pg/ml, such as in the range of 0.15- 5 pg/ml, e.g. in the range of 0.2-2 pg/ml, such as in the range of 0.25-1 pg/ml, e.g. in the range of 0.3-0.75 pg/ml, such as in the range of 0.035-0.5 pg/ml, e.g. in the range of 0.01-0.1 pg/ml, such as in the range of 0.025-0.05 pg/ml