FIELD

[001] Embodiments of the present disclosure relate generally to methods for killing persister cells or inhibiting the growth of persister ceils and methods for treating and/or preventing conditions, diseases and disorders associated with persister cells.

BACKGROUND

[002] Microbial persister cells are a subpopulation of cells which possess characteristics distinct to those of their wild type counterparts. Persister ceils have been identified in a wide range of genera and species, including, but not limited to, Staphylococcus spp., such as S. aureus, S. epidermidis, and .V. capitis; Pseudomonas spp. such as P. aeruginosa, Burkholderia spp. such as B. cepacia and B. pseudomallei; Salmonella serovars, including Salmonella Typhi; Vibrio spp. such as V. cholerae; Shigella spp., Brucella spp. such as B. melitensis; Escherichia spp. such as E. coli; Lactobacillus spp. such as L acidophilus; Serratia spp. such as S. marcescens; Neisseria spp. such as N. gonorrhoeae, as well as the unicellular fungus Candida albicans.

[003] Persister ceils are characterized by a slow-growing phenotype; that is, persister cells divide at a significantly slower rate than the rate in logarithmic bacterial growth. Indeed, some persister cells are "dormant"; that is, the cells are not undergoing cell division. In these dormant persister cells, cellular metabolism is reduced even further than in slow-growing cells, leading to the ceils being sometimes described as

'metabolically inactive' or 'non-growing', despite the fact that dormant persister ceils, like all living cells, require some form of cellular energy and redox homeostasis to maintain cell viability, even in the absence of growth (i.e. no cell division).

[004] Persister cells are significant in microbial pathogenicity since they are often highly resistant to antimicrobials; they are particularly associated with latent and recurrent infections. The basis for persister cell antimicrobial resistance is

multifactorial . For example, the slow-rate (or absence) of cell division in persister cells means that many of the biosynthetic pathways targeted by known antibiotics are not in active use, making the persister cell 'indifferent' to the effects of the antibiotic. In addition to this, persister cells are often able to persist inside host cells, likely contributing to the persister cell's resistance to antimicrobials. These properties, together with the fact that persister cells have been isolated from abscesses, blood, the respiratory tract, soft tissues, bones, and joints of patients, mean that persister ceils are clinically important microorganisms.

[005] Staphylococcal persister cells have been associated with a wide range of infections and conditions. Staphylococcal persister cells include small colony variants (SCVs), originally named because the colonies are typically approximately one-tenth the size of their wild-type counterparts as a result of their slow growth rate. Of particular clinical concern are S. aureus persister cells, which display increased resistance to some antibiotics such as aminoglycosides (e.g. gentamicin) compared to parental strains. S. aureus persister cells have been associated with numerous persistent and recurrent S. aureus infections, including, but not limited to, respiratory infections in cystic fibrosis subjects, osteomyelitis, skin infections and infections associated with medical implants. Although S. aureus is normally an extracellular organism, S. aureus persister cells can reside intracellularly, thus contributing to their ability to cause persistent and recurrent infections.

[006] Staphylococcal persister cells, such as S. aureus persister cells, are typically associated with auxotrophism for hemin, thiamine or menadione, which is related to mutations in genes encoding products involved in the electron transport system, such as the menD, hemB, and ctaA genes. Other phenotypic characteristics of these auxotrophic S. aureus persister cells can include, but are not limited to, reduced pigment formation, reduced hemolytic activity, descreased coaguiase activity. In some instances, S. aureus persister cells are thymidine-dependent, having a defect in their thymidine biosynthetic pathway. Such persister cells are particularly associated with long term trimethoprim sulphamethoxazole (SXT) treatments of patients with cystic fibrosis, wherein thymidine-dependent, SXT-resistant persister cells emerge in the respiratory tract after SXT treatment to suppress infections in these patients.

[007] The clinical importance of persister cells means that there is a need for improved methods for killing persister ceils or inhibiting the growth of persister cells and for treating and/or preventing conditions, diseases and disorders associated with persister cells.

SUMMARY

[008] The present disclosure provides methods for killing persister ceils or inhibiting the growth of a microbial persister cell, comprising exposing the persister cell to an effective amount of auranofm. The methods may be performed, for example, in vivo, ex vivo, or in vitro.

[009] The present disclosure also provides methods for treating or preventing an infection, disease or disorder caused by or associated with a microbial persister ceil in a subject, comprising administering to the subject an effective amount of auranofm. The methods may be performed, for example, in vivo, ex vivo, or in vitro.

[0010] The present inventors have observed that auranofm is able to inhibit the growth of, or kill, microbial persister cells which are resistant to other antimicrobial agents. Accordingly, the present disclosure provides methods for reducing the number, density or proportion of persister cells in a microbial population, the method comprising exposing the persister cell to an effective amount of a compound of the disclosure. In some embodiments the number, density or proportion of persister cells in a microbial population is reduced by at least 10% compared to an otherwise identical population not exposed to auranofm; for example, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%*, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.9%, or at least 99.99%. The methods may be performed, for example, in vivo, ex vivo, or in vitro.

[0011] Unlike many other antimicrobial agents which are unable to inhibit the growth of or kill microbial persister cells, auranofm is able to inhibit the growth of or kill microbial persister cells at a similar efficacy to non-persister cells. Accordingly, the present disclosure provides methods for inhibiting the growth of, reducing the size of, or eliminating a population of microbes comprising microbial persister cells, the method comprising exposing the population to an effective amount of auranofm. In some embodiments the size of the population of microbes comprising microbial persister cells is reduced by at least 10% compared to an otherwise identical population not exposed to auranofm; for example, at least 20%, at least 30%, at least 40%>, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%, The methods may be performed, for example, in vivo, ex vivo, or in vitro.

[0012] The present disclosure also provides methods of preventing the formation of microbial persister cells in a microbial population, the method comprising exposing the population to an effective amount of auranofin.

[0013] In particular embodiments of the methods of the present disclosure, the persister cell is a bacterial or fungal persister cell. In some examples, the persister cell is a Gram negative bacterium. In some examples, the persister cell is a Gram positive bacterium. In some examples, the persister cell is a small colony variant. In some aspects of the methods of the present disclosure, the persister cell is a Staphylococcus spp. persister cell, such as, for example, a S. aureus, S. epidermidis or S. capitis persister ceil. In one example, the persister cell is a S. aureus small colony variant. In other aspects, the persister cell is a Pseudomonas spp, Burkholderia spp, Salmonella, serovars, Vibrio spp., Shigella spp., Brucella, spp., Escherichia spp, Serratia spp., Neisseria spp. or Candida spp. persister cell,

[0014] The methods of the present disclosure may also include administering at least one additional antimicrobial agent, such as, for example, an antibiotic or antifungal agent.

[0015] The present disclosure is also directed to use of auranofin for the preparation of a medicament for killing a microbial persister cell, or inhibiting the growth of a microbial persister ceil.

[0016] The present disclosure is also directed to use of auranofin for the preparation of a medicament for treating or preventing an infection, disease or disorder caused by or associated with a microbial persister cell. The use may be, for example, in vivo, ex vivo, or in vitro.

[0017] In some embodiments of the uses of the present disclosure, the persister cell is a bacterial or fungal persister cell. In some examples, the persister cell is a Gram negative bacterium. In some examples, the persister cell is a Gram positive bacterium. In particular aspects, the persister cell is a small colony variant. In some examples, the persister cell is a Staphylococcus spp. persister cell, such as, for example, a S. aureus, S. epidermidis or S. capitis persister cell. In one example, the persister ceil is a S. aureus small colony variant. In further examples, the persister ceil is a Pseudomonas spp, Burkholderia spp, Salmonella, serovars, Vibrio spp., Shigella spp., Brucella, spp., Escherichia spp, Serratia spp., Neisseria spp. or Candida spp. persister cell.

[0018] Also provided is auranofin, or compositions comprising auranofin, for use in a method of treatment or prevention of an infection, disease or disorder caused by or associated with a microbial persister cell. In some aspects, the persister cell is a bacterial or fungal persister cell. In some examples, the persister cell is a Gram negative bacterium. In some examples, the persister cel l is a Gram positive bacterium. In particular examples, the persister cell is a small colony variant. In some embodiments, the persister ceil is a Staphylococcus spp. persister cell (e.g. a S. aureus, S. epidermidis or S. capitis persister cell). In one aspect, the persister cell is a S. aureus small colony variant. In further aspects, the persister cell is a Pseudomonas spp, Burkholderia spp, Salmonella, serovars, Vibrio spp,, Shigella spp., Brucella, spp., Escherichia spp, Serratia spp., Neisseria spp, or Candida spp, persister cell. The compositions of the present disclosure may also comprise at least one additional antimicrobial agent. For example, the compositions can comprise an antibiotic or antifungal agent.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Embodiments of the disclosure are described herein, by way of non-limiting example only, with reference to the following figures.

[0020] Figure 1 shows growth inhibition of various S. aureus strains in Cation- Adjusted Mueller-Hinton Broth (CaMHB) in the presence of various concentrations of auranofin, expressed as a percentage of the growth (as determined by absorbance at 595 ran) of the same strains in the absence of auranofin. A. Growth inhibition of S. aureus NCTC 8325. B. Growth inhibition of S. aureus clinical isolates 04-229-2455, 04-228- 1825, 04-227-3567 and 02-228-361 1 , and hospital and community acquired MRSA strains IMVS67 (nmMRSA D), RBH98 (QLD), PAHS 8 (SWP), MW2 (USA400), CH I 6 (UK EMRSA-15) RPAH18 (Aus-2) and US A300,

[0021] Figure 2 represents the results of a time kill assay to assess the effect of auranofin on the growth of S. aureus. The number of colony forming units (cfu)/mL was determined at various intervals during culture of S. aureus in the presence of auranofm, rifampicin, or a combination of auranofin and rifampicin (triangles, 0.5 _, ug/mL auranofin, filled squares, 1 ^ig/mL auranofm; small circles, 5 g/mL auranofin; inverted triangles, 0.2 μg/mL rifampicin; diamonds, 1 μg/mL rifampicin; large circles, 0.5 μg/mL auranofm and rifampicin). A control in which S. aureus was cultured in the absence of either auranofm or rifampicin was also included (open squares).

[0022] Figure 3 shows the effective treatment of S. aureus invasion of THP-1 cells with auranofm. THP-1 cells were infected with a moi~3 of S. aureus SHIOOOgfpfor 1.5 hours prior to addition of auranofm, pt = ^;: post treatment.

[0023] Figure 4 shows the growth inhibition of a S. aureus perister cell (SCV - hemB mutant) (circles) and the parental NCTC 8325 strain (squares) in the presence of varying concentrations of gentamicin (A) and auranofin (B).

[0024] Figure 5 is a photograph showing plate growth of S. aureus NCTC 8325 (top plates) and a SCV (hemB mutant) thereof (bottom plates) in the presence of 4 ^ig auranofin and 100 ,ug gentamicin (far left), 100 ,ug gentamicin alone (middle) and 4 μg auranofin alone (right).

[0025] Figure 6 shows the growth of S. aureus NCTC8325 in the supernatant of A549 cells (adenocarcinoma human alveolar basal epithelial cells) or intracellularlv, in the presence or absence of I μg mL auranofm (Au) or 100 units/mL penicillin and 100 μg/mL streptomycin (P/S).

[0026] Figure 7 shows the growth of S. aureus NCTC8325.4 in the presence or absence of 1 μ ½ί auranofm (Au), 1 ug/mL auranofin and 0.2 μg/mL rifampicin (Au/rif), 0.2 ug/mL rifampicin (rif), or 100 units/mL penicillin and 100 μg/mL streptomycin (P/S), in the supernatant cells of A549 cells (A) or intracellularlv (B and C) at various time points.

[0027] Figure 8 A & 8B shows the results of two resistance frequency assays (assay 1 [8 A] and assay 2 [8B]) to assess whether auranofm -resistant S. aureus mutants developed. S. aureus was streaked out on CaMHB agar plates containing l , 1.5x, 2x and 4x the minimum inhibitory concentration of auranofm. Any potential mutants were picked and analysed. S. aureus was also grown on TSA plates and disc-diffusion assays carried out using standard conditions (with discs impregnated with either rifampicin or auranofm). Rifampicin-resistant (rif resist) cells were picked from the rifampicin zone of inhibition and potential auranofin-resistant (Au resist) mutants were picked from the edge of the auranofin zone of inhibition and cultured in CaMHB in the presence or absence of auranofin, rifampicin or lincomycin. Bacteria that had not originally been grown in the agar plates and which were not resistant (wt) were also included in the growth assay. The growth of the bacteria was assessed and expressed as a percentage of the control (i.e. bacteria grown in the absence of antibiotic).

[0028] Figure 9 shows the sensitivity to auranofin of a range of redox mutants.

DETAILED DESCRIPTION

The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an antimicrobial agent" means one antimicrobial agent or more than one antimicrobial agent.

[0029] In the context of this specification, the term "about" is understood to refer to a range of numbers that a person of skill in the art would consider equivalent to the recited value in the context of achieving the same function or result.

[0030] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0031] As used herein, "persister cell" refers to metabolic variants of wild type microbial cells that are phenotypically characterized by their slow growth rate, which is typically 30%, 25%, 20%, 15%, 10%, 5% or less of the growth rate of the wild-type counterpart. In some embodiments, the persister cells are dormant and have, for example, no detectable cell division in a 24 hour period. Further, persister cells typically form colonies that are approximately 30%, 25%, 20%, 15%, 10%, 5% or less of the size of the colonies formed by their wild-type counterparts. Reference to persister cells includes reference to persister cells of any microbial genera or species, including, but not limited to, bacterial and lower eukaryotic, such as fungal, including yeast, persister cells. In some examples, the persister cell is a Gram negative bacterium. In some examples, the persister cell is a Gram positive bacterium. Exemplar}' persister cells include, but are not limited to, those of Staphylococcus spp., such as S. aureus, S. epidermidis, and S. capitis; Pseudomonas spp. such as P. aeruginosa; Burkholderia spp. such as B. cepacia and />'. pseudomallei; Salmonella serovars, including Salmonella Typhi; Vibrio spp. such as V. cholerae; Shigella spp.; Brucella spp. such as /)'. melitensis, Escherichia spp. such as E. coli; Lactobacillus spp. such as L. acidophilus; Serratia spp. such as S. marcescens; Neisseria spp. such as N. gonorrhoeae, as well as Candida spp., such as C. albicans.

[0032] As used herein the term "antimicrobial agent" refers to any agent that, alone or in combination with another agent, is capable of killing or inhibiting the growth of one or more species of microorganism. Antimicrobial agents include, but are not limited to, antibiotics, antifungals, detergents, surfactants, agents that induce oxidative stress, bacteriocins and antimicrobial enzymes (e.g. lipases, pronases and lyases) and various other proteolytic enzymes and nucleases, peptides and phage. Reference to an antimicrobial agent includes reference to both natural and synthetic antimicrobial agents.

[0033] As used herein the term "effective amount" includes within its meaning a nontoxic but sufficient amount of an agent to provide the desired effect. The exact amount required will vary from subject to subject or situation to situation depending on factors such as the species of bacteria being exposed to the agent (e.g. auranofin), the severity of the disease or disorder associated with the bacteria, the mode of administration of the agent and so forth. Thus, it is not possible to specify an exact "effective amount". However, for any given case, an appropriate "effective amount" may be determined by one of ordinary skill in the art using only routine experimentation.

[0034] As used herein the term "exposing" means generally bringing into contact with. Exposure of a persister cell to an agent (e.g. auranofin) includes administration of the agent to a subject harboring the microorganism, or otherwise bringing the microorganism into contact with the agent itself, such as by contacting a surface on which the microorganism is present with the agent. In the present disclosure the terms "exposing", "administering" and "contacting" and variations thereof may, in some contexts, be used interchangeably. [0035] The term "inhibiting" and variations thereof such as "inhibition" and "inhibits" as used herein in relation to growth of a microorgani sm refers to any microbicidal or microbiostatic activity of an agent (e.g. auranofin) or composition. Such inhibition may ^¬ be in magnitude and/or be temporal or spatial in nature. Inhibition of the growth of a microorganism by an agent can be assessed by measuring growth of the microorganism in the presence and absence of the agent. The growth can be inhibited by the agent by at least or about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more compared to the growth of the same microorganism that is not exposed to the agent.

[0036] As used herein the terms "treating", "treatment", "preventing" and "prevention" refer to any and all uses which remedy a condition or symptoms, prevent the establishment of an infection, condition or disease, or otherwise prevent, hinder, retard, or reverse the progression of an infection, condition or disease or other undesirable symptoms in any way whatsoever. Thus the terms "treating" and "preventing" and the like are to be considered in their broadest context. For example, treatment does not necessarily imply that a patient is treated until total recover}' .

[0037] As used herein, a "subject" includes human and non-human animals, including, for example, livestock (such as cows, sheep, goats, pigs horses and goats), companion animals (such as cats and dogs) and show or performance animals (such as horses).

[0038] In part, embodiments of the present disclosure relate generally to the use of auranofin in methods for killing persister cells or inhibiting the growth of persister cells and methods for the treatment or prevention of infections, diseases and disorders caused by or associated with persister cells, comprising exposing the persister cells to the auranofin.

(I)

[0039] Auranofin (2,3 ,4,6-tetra-o-acetyl- 1 -thio-P-D-glucopyranosato-S-(triethyl- phosphine) gold, or triethylphosphine gold thioglucose tetra-acetate) is a gold derivative with a molecular weight of 678.5 and having the structure

Marketed as Ridaura®, auranofin is an anti-arthritic agent used in the treatment of rheumatoid and juvenile arthritis. Auranofin has been shown to inhibit leukocyte activation pathways at multiple sites, as well as inhibit the release of inflammatory mediators from human basophils, pulmonary mast cells and macrophages. Auranofin is thought to act as an anti-arthritic because of its inhibitor}' effect on a number of proteases involved in the progression of rheumatoid arthritis, including its strong inhibition of the selenoenzyme thioredoxin reductase (TrxR), both in the cytosol and in the mitochondria.

[0040] More recently, auranofin has been shown to restrict the viral reservoir in HIV- infected monkeys, inhibit the growth of the parasites Trypanosona brucei and Schistosoma mansoni, and inhibit the growth of Enierococcus faecalis and Clostridium difficile. In some studies in which the anti -bacterial effect of auranofin was investigated, results indicated that auranofin blocks or disrupts selenium metabolism in these selenium-dependent organisms by forming a complex with selenium, which in turn prevents selenium-uptake and incorporation into enzymes.

[0041] The present disclosure provides methods for inhibiting the growth of microbial persister cells, by exposing the persister ceils to an effective amount of auranofin. Exposure can be achieved by applying the auranofin to the surface of an object or to an internal or external bodily surface. In some aspects of the present disclosure, the persister cells are small colony variants (SCVs). In particular embodiments, the persister cells are Staphylococcus spp. (including Staphylococcal SCVs), such as S. aureus (including methicillin resistant S. aureus (MRSA)), .V. epidermidis, and S. capitis. In further embodiments, the persister cells are Pseudonionas spp. such as P. aeruginosa; Burkholderia spp. such as B. cepacia and B. pseudornallei; Salmonella serovars, including Salmonella Typhi; Vibrio spp. such as V. cholerae; Shigella spp.; Brucella spp. such as B. melitensis, Escherichia spp. such as ; ^' . coli, Lactobacillus spp. such as L. acidophilus; Serratia spp. such as S. marcescens; Neisseria spp. such as N. gonorrhoeae, or Candida spp., such as C. albicans. The compound of formula I, such as auranofin, or a derivative or analogue thereof may be microbicidal or microbiostatic against the persister cells. Also provided are compositions comprising auranofin for use in such methods.

[0042] Without wishing to be bound by theory, it appears that Auranofin exerts its effect on persister cells at least partially through a link with impaired redox-pathway activity and enhanced oxidative stress. In particular, it appears that Auranofin inhibits the bacterial thioredoxin (Trx) pathway (see Example 5). This is consistent with Auranofin' s believed mechanism of action in Rheumatoid arthritis, where it inhibits the selenoenzvme thioredoxin reductase (TrxR), both in the cvtosol and in the mitochondria (ibid.).

[0043] In particular aspects, the present disclosure is directed to the use of auranofin to inhibit the growth of staphylococcal persister cells, such as S. aureus (including MRSA), S. epidermidis, and S. capitis persister cells. In one embodiment, the methods provided herein are directed to inhibiting the growth of S. aureus persister cells by exposing the persister cells to an effective amount of the auranofin. As demonstrated herein, auranofin inhibits the growth of S. aureus persister cells. Furthermore, auranofin is effective against both intracellular and extracellular microbial cells, even in the presence of serum, properties that are important for clinical use.

[0044] Persister cells, such as Staphylococcal persister ceils, are known to cause infections associated with the implantation of medical devices including, but are not limited to, venous catheters, drainage catheters (e.g. urinary catheters), stents, pacemakers, contact lenses, hearing-aids, percutaneous glucose sensors, dialysis equipment, drug-pump related delivery cannula, prostheses such as artificial joints, hearts, heart valves or other organs, medical fixation devices (e.g. rods, screws, pins, plates and the like), or devices for wound repair, such as sutures and wound dressings such as bandages. Accordingly, in some aspects the methods of present disclosure include coating, impregnating or otherwise contacting such devices with auranofm thereby exposing the persister cells to the auranofin and killing the persister cells, or inhibiting their growth. In a related aspect of the disclosure, a medical device (such as those exemplified above) coated or impregnated with a compound of the disclosure is provided.

[0045] A further embodiment of the disclosure is a method for treating or preventing an infection, disease or disorder caused by or associated with persister cells in a subject, comprising administering to the subject an effective amount of auranofm. Also provided are compositions comprising auranofm for use in such methods. Subjects thai- are particularly susceptible to persister ceil infection include, for example, subjects with cystic fibrosis. Persister cells such as S. aureus, P. aeruginosa and B. cepacia persister cells can be the cause of respiratoiy tract infection and sepsis in subjects with cystic fibrosis. Other infections, diseases or disorders associated with persister cells and thai- can be treated according to the present disclosure include, but are not limited to, osteomyelitis (e.g. Staphylococcal persister cells), endocarditis (e.g. B. melitensis persister cells), urinary tract infection (e.g. E. coli persister cells), gonorrhea (e.g. N. gonorrhoeae persister cells), typhoid fever (Salmonella serovar persister ceils), and infections associates with surgical procedures and medical implants (e.g. Staphylococcal and E. coli persister cells).

[0046] In particular embodiments, the present disclosure is directed to methods for treating or preventing an infection, disease or disorder caused by or associated with S. aureus persister cells, comprising administering to the subject an effective amount of auranofin. Exemplar}' infections, diseases or disorders caused by S. aureus persister cells include, but are not limited to, skin infections, osteomyelitis, pulmonary infections (including pulmonary infection in patients with cystic fibrosis), peritonitis, pneumonia, tympanitis, sinusitis, cellulitis, impetigo, mastitis, bacterial endocarditis, sepsis, urinary tract infections, dental plaque, dental caries, periodontitis, bacterial prostatitis, and infections associated with surgical procedures or bums. Accordingly, provided are methods for treating or preventing a skin infection, osteomyelitis, pulmonary infection (including pulmonary infection in patients with cystic fibrosis), peritonitis, pneumonia, tympanitis, sinusitis, cellulitis, impetigo, mastitis, bacterial endocarditis, sepsis, urinary tract infection, dental plaque, dental caries, periodontitis, bacterial prostatitis, and infections associated with surgical procedures, medical implants or burns, caused by S. aureus persister cells, comprising administering to the subject an effective amount of auranofin.

[0047] In the methods and uses of the present disclosure the auranofin can be administered to the subject by any route understood to be suitable by a skilled artisan. Non-limiting examples of suitable routes of administration include intranasal, oral, intraarterial, intravenous (including by discrete injection, intravenous bolus or continuous infusion), intramuscular, intradermal, transdermal, subcutaneous, intraperitoneal or topical administration (including topical administration to the eye) as well as any combination of any two or more of these routes. Auranofin can be administered to a subject once or more than once, including 3, 4, 5, 6 or more times, or as many times as required to achieve the desired outcome (e.g. control of the infection), and at any appropriate interval.

[0048] In particular embodiments, the auranofin is used in combination with one or more other antimicrobial agents. Accordingly, provided are methods for killing microbial persister cells, or inhibiting the growth of persister cells, by exposing the persister cells to an effective amount of the auranofin and one or more other antimicrobial agents. Also provided are methods for the treatment or prevention of an infection, disease or disorder caused by or associated with persister cells, comprising administering auranofin and one or more other antimicrobial agents to the subject.

[0049] The auranofin and the one or more other antimicrobial agents can be administered at the same time or at different times, i.e. exposure can be simultaneous or sequential. Furthermore, they can be administered or delivered by the same or different routes or means. For example, where the auranofin and the one or more other antimicrobial agents are administered to a subject, they can be co-formulated in the same composition or formulated in different compositions and administered by the same route or different routes, e.g. by intranasal, oral, intraarterial, intravenous (including by discrete injection, intravenous bolus or continuous infusion), intramuscular, intradermal, transdermal, subcutaneous, intraperitoneal or topical administration, simultaneously or sequentially. [0050] Exemplar}' antimicrobial agents suitable for the methods described herein include, but are not limited to, antibiotics, antifungals, detergents, surfactants, agents that induce oxidative stress, bacteriocins and antimicrobial enzymes (e.g. lipases, pronases and lyases) and various other proteolytic enzymes and nucleases, peptides and phage. The antimicrobial agents may be natural or synthetic. The antimicrobial agent employed may be selected for the particular application of the disclosure on a case-by- case basis, and those skilled in the art will appreciate that the scope of the present disclosure is not limited by the nature or identity of the particular antimicrobial agent.

[0051] Non-limiting examples of antimicrobial agents include fluoroquinolones, aminoglycosides, glycopeptides, lincosamides, cephalosporins and related beta-lactams, macrolides, nitroimidazoles, penicillins, polymyxins, tetracyclines, and any combination thereof. For example, the methods of the present disclosure can employ acedapsone; acetosulfone sodium; alamecin; alexidine; amdinocillin; amdinocillin pivoxil; amicycline; amifloxacin; amifloxacin mesylate; amikacin; amikacin sulfate; aminosalicylic acid; aminosalicylate sodium; amoxicillin, amphomycin; ampicillin; ampicillin sodium; apalcillin sodium, apramycin; aspartocin; astromicin sulfate; avilamycin; avoparcin; azithromycin; aziocillin; azlocillin sodium; bacampiciliin hydrochloride; bacitracin; bacitracin methylene disalicylate; bacitracin zinc; bambermycins; benzoyipas calcium; berythromycin; betamicin sulfate; biapenem; biniramycin; biphenamine hydrochloride; bispynthione magsulfex; butikacin; butirosin sulfate; capreomycin sulfate; carbadox; carbenicillin disodium; carbenicillin indanyl sodium, carbenicillin phenyl sodium, carbenicillin potassium; carumonam sodium; cefaclor; cefadroxii; cefamandole; cefamandole nafate; cefamandole sodium; cefaparole; cefatrizine; cefazaflur sodium; cefazolin; cefazolin sodium; cefbuperazone; cefdinir; cefepime; cefepime hydrochloride, cefetecol ; cefixime; cefmenoxime hydrochloride; cefmetazole; cefmetazole sodium; cefonicid monosodium; cefonicid sodium, cefoperazone sodium; ceforanide; cefotaxime sodium; cefotetan, cefotetan disodium; cefotiam hydrochloride; cefoxitin; cefoxitin sodium; cefpimizole; cefpimizole sodium, cefpiramide; cefpiramide sodium; cefpirome sulfate, cefpodoxime proxetil; cefprozii; cefroxadine; cefsulodin sodium; ceftazidime; ceftibuten; ceftizoxime sodium, ceftriaxone sodium; cefuroxime; cefuroxime axetil; cefuroxime pivoxetil; cefuroxime sodium; cephacetrile sodium; cephalexin; cephalexin hydrochloride; cephaloglycin; cephaloridine; cephalothin sodium; cephapirin sodium; cephradine; cetocycline hydrochloride, cetophenicol; chloramphenicol; chloramphenicol palmitate; chloramphenicol pantothenate complex; chloramphenicol sodium succinate; chlorhexidine phosphani late; chloroxylenol; chlortetracycline bi sulfate; chlortetracycline hydrochloride; cinoxacin; ciprofloxacin; ciprofloxacin hydrochloride; cirolemycin; clarithromycin, clinafloxacin hydrochloride, clindamycin; clindamycin hydrochloride; clindamycin palmitate hydrochloride; clindamycin phosphate; clofazimine, cloxacillin benzathine, cloxacillin sodium, chlorhexidine, cloxyquin; colistimethate sodium; colistin sulfate; coumermycin; coumermycin sodium; cyclacillin; cycloserine; dalfopristin; dapsone; daptomycin; demeclocycline; demeclocycline hydrochloride; demecycline; denofungin; diaveridine; di cloxacillin, dicloxacil lin sodium; dihydrostreptomycin sulfate; dipyrithione; dirithromycin; doxycycline; doxycycline calcium; doxycycline fosfatex; doxycycline hy elate; droxacin sodium; enoxacin; epiciliin; epitetracycline hydrochloride; erythromycin; erythromycin acistrate; erythromycin estolate; erythromycin ethylsuccinate; erythromycin gluceptate; erythromycin lactobionate; erythromycin propionate; erythromycin stearate; ethambutol hydrochloride; ethionamide; fleroxaein; fl oxacillin; fludalanine; flum equine; fosfomycin; fosfomycin tromethamine; fumoxicillin; furazoiium chloride; furazolium tartrate; fusidate sodium, fusidic acid; ganciclovir and ganciclovir sodium; gentamicin sulfate; gloximonam ; gramicidin; haloprogin; hetacillin; hetacillin potassium; hexedine; ibafloxaein; imipenem; isoconazole; isepamicin; isoniazid; josamycin; kanamycin sulfate; kitasamycin; levofuraltadone; levopropylcillin potassium; lexithromycin; lincomycin; lincomycin hydrochloride; lomefloxacin; lomefloxacin hydrochloride; lomefloxacin mesylate, loracarbef, mafenide; meclocycline; meclocycline sulfosalicylate; megalomicin potassium phosphate; mequidox; meropenem; methacycline, methacycline hydrochloride; methenamine; methenamine hippurate; methenamine mandelate; methiciliin sodium; metioprim; metronidazole hydrochloride; metronidazole phosphate, mezlocillin; mezlocillin sodium; minocycline, minocycline hydrochloride; mirincamycin hydrochloride; monensin; monensin sodiumr; nafcillin sodium; nalidixate sodium; nalidixic acid; natainycin; nebramycin; neomycin palmitate; neomycin sulfate; neomycin undecylenate; netilmicin sulfate; neutramycin; nifuiradene; nifuraldezone; nifuratel; nifuratrone; nifurdazil; nifurimide; nifiupirinoi; nifurquinazol; nifurthiazole; nitrocycline; nitrofurantoin, nitromide; norfloxacin; novobiocin sodium; ofloxacin; onnetoprim; oxacillin and oxacillin sodium, oximonam; oximonam sodium; oxoiinic acid; oxytetracycline; oxytetracycline calcium; oxytetracycline hydrochloride; paldimycin; parachlorophenol; paulomycin; pef!oxacin; pefloxacin mesylate; penamecillin; penicillins such as penicillin G benzathine, penicillin G potassium, penicillin G procaine, penicillin G sodium, penicillin V, penici llin V benzathine, penicillin V hydrabamine, and penicillin V potassium; pentizidone sodium; phenyl aminosalicylate; piperacillin sodium, pirbenicillin sodium; piridicillin sodium; pirlimycin hydrochloride; pivampicillin hydrochloride; pivampicillin pamoate; pivampicillin probenate; polymyxin b sulfate; porfiromycin; propikacin; pyrazinamide; pyrithione zinc; quindecamine acetate; quinupristin; racephenicol; ramoplanin; ranimycin; relomycin; repromicin; rifabutin; rifametane; rifamexil; rifamide; rifampin; rifapentine; rifaximin; rolitetracycline; roli tetracycline nitrate; rosaramicin; rosaramicin butyrate; rosaramicin propionate; rosaramicin sodium phosphate; rosaramicin stearate; rosoxacin; roxarsone; roxithromycin; sancycline, sanfetrinem sodium; sarmoxicillin; sarpicillin; scopafungin; sisomicin; sisomicin sulfate; sparfloxacin; spectinomycin hydrochloride; spiramycin; stallimycin hydrochloride; steffimycin; streptomycin sulfate; streptonicozid; sulfabenz; sulfabenzamide; sulfacetamide; sulfacetamide sodium; sulfacytine; sulfadiazine; sulfadiazine sodium, sulfadoxine; sulfalene; sulfamerazine; sulfameter; sulfamethazine; sulfamethizole; sulfamethoxazole; sulfamonomethoxine; suifamoxole; sulfanilate zinc; sulfanitran; sulfasalazine; sulfasomizole; sulfathiazole; sulfazamet; sulfisoxazole; sulfi soxazole acetyl; sulfisboxazole diolamine; sulfomyxin; sulopenem; sultamriciliin; sunciliin sodium; talampicillin hydrochloride; teicoplanin, temafloxacin hydrochloride; temocillin; tetracycline; tetracycline hydrochloride; tetracycline phosphate complex; tetroxoprim; thiamphenicol; thiphencillin potassium, ticarcillin cresyl sodium; ticarcillin disodium; ticarcillin monosodium; ticlatone; tiodonium chloride; tobramycin; tobramycin sulfate; tosufloxacin, trimethoprim; trimethoprim sulfate; trisulfapyrimidines; troleandomycin; trospectomycin sulfate; tyrothricin; vancomycin; vancomycin hydrochloride; virginiamycin; zorbamycin; imidazoles (e.g. bifonazolem butoconazoie, clotrimazole, econazole, fenticonazole, isoconazole, ketoconazole, miconazole, omoconazole, oxiconazole, sertaconazole, sulconazole, tioconazoie, triazoles (e.g. albaconazoie, fluconazole, isavuconazole, itraconazole, posaconazole, ravuconazole, terconazole, voriconazole), thiazoles (e.g. abafungin), ally! amines (e.g. amorolfm, butenafine, naftifine, terbinafme) and echinocandins (anidulafungin, caspofungin, micafungin) and combinations thereof,

[0052] In particular examples, antimicrobial agents suitable for use in combination with auranofm to treat infections, diseases or disorders associated with or caused by microbial persister cells, including S. aureus persister cells, may include, but are not limited to, methicillin, nafciilin, oxacillin, cloxaciliin, dicloxacillin, flucloxacillin, clindamycin, co-trimoxazole, rifampicin, lincomycin, vancomycin, teicoplanin and mupirocin.

[0053] The auranofm can be formulated as a composition in a manner suitable for the desired application. Accordingly, provided are compositions comprising auranofm for use in the methods of the present disclosure. The auranofm can be produced by any method known in the art. For example, auranofm may be produced using methods described in U.S. Patent. Nos. 41 15642, 4122254, 4125710, 4125711, 4131732, 4133952 and 4200738.

[0054] The particular formulation of the composition comprising auranofm will depend on the application or delivery method to the required surface and thus will vary with different applications. For example the composition may be formulated for particular routes of administration, such as in the form of a liquid, suspension, syrup, nasal spray (including in a form for administration using a nebulizer), eyedrops, powder, tablet, capsule, cream, paste, gel or lotion. In further examples, auranofm is formulated for controlled release. The skilled addressee will recognize that the appropriate formulation will depend on the particular application and the proposed route of delivery.

[0055] For in vivo administration to a subject, compositions comprising auranofm may include one or more pharmaceutically acceptable earners, excipients or diluents. Examples of pharmaceutically acceptable carriers, excipients or diluents are demineralised or distilled water, saline solution; vegetable based oils such as peanut oil, saffiower oil, olive oil, cottonseed oil, maize oil, sesame oil, arachis oil or coconut oil; silicone oils, including polysiloxanes, such as methyl poiysiioxane, phenyl polysiloxane and methylphenyl polysolpoxane; volatile silicones, mineral oils such as liquid paraffin, soft paraffin or squalane; cellulose; cellulose derivatives such as methyl cellulose, ethyl cellulose, carboxymethylcel lulose, sodium carboxymethyl cellulose or hydroxypropylmethylceliulose; lower alkanois, for example ethanol or iso-propanol; lower aralkanols; lower polyalkylene glycols or lower alkylene glycols, for example polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1,3- butylene glycol or glycerin; fatty acid esters such as isopropyl palmitate, isopropyl myri state or ethyl oleate; polyvinylpyrridone; agar; carrageenan; gum tragacanth or gum acacia, and petroleum jelly.

[0056] The compositions are formulated with an amount or concentration of auranofm that is suitable for use in the particular embodiment of the disclosure, e.g. at concentrations or amounts sufficient to kill the microbial persister cells, or inhibit the growth of the persister cells, and/or prevent and/or treat the condition, disease or disorder associated with the persister cells. The compositions can be formulated for direct administration, or can be formulated as a concentrated composition that is subsequently diluted prior to use. In some instances, the compositions are in solid form, such as in tablet or capsule form, and contain auranofm at a concentration of between about 0.01% (w/w) and about 50% (w/w), between about 0.1% (w/w) and about 20% (w/w), between about 0.5% (w/w) and about 10% (w/w), or between about 1% (w/w) and about 5% (w/w). In other instances, the compositions are in liquid form. In particular embodiments, such compositions are formulated with between about 1 ng/mL and about 100 mg/mL, between about 10 ng/mL and about 100 mg/mL, between about 100 ng/mL and about 1 100 mg/mL, between about 1 ug/mL and about 10 mg/mL, between about 10 μg/mL and about 10 mg/mL, between about 100 μg/mL and about 10 mg/mL, or between about I mg/mL and about 10 mg/mL auranofm. The most suitable concentration to achieve the desired effect wil l depend on a number of factors and may be determined by those skilled in the art using routine experimentation.

[0057] In embodiments where auranofm is utilized in accordance with the present disclosure with one or more other antimicrobial agents, the auranofm and one or more other antimicrobial agents can be co-formulated or formulated in separate compositions. In instances where the agents are formulated in different compositions, they can be administered or delivered by the same or different routes or means and thus forumulated accordingly. For example, where auranofm and the one or more other antimicrobial agents are administered to a subject, they can be co-formulated in the same composition or formulated in different compositions and administered by the same route or different routes, e.g. by intranasal, oral, intraarterial, intravenous (including by discrete injection, intravenous bolus or continuous infusion), intramuscular, intradermal, transdermal, subcutaneous, intraperitoneal or topical administration, simultaneously or sequentially.

[0058] The compositions can be administered to a subject in therapeutically effective amounts (e.g., amounts that prevent or reduce progression of a disease or condition) to provide therapy for the disease or condition. The precise amount or dose of the auranofin that i s administered to the subject depends on several factors, including, but not limited to, the severity of the disease or condition, the use of other antimicrobial agents, the route of administration, the number of dosages administered, and other considerations, such as the weight, age and general state of the subject. Particular dosages can be empirically determined or extrapolated from, for example, studies in animal models or previous studies in humans. For example, in some embodiments, auranofin may be administrated to a subject at a dose of between about 0.05 mg/kg body weight and about 100 mg/kg, such as at least or about 0.05 mg/kg, 0.1 mg/kg, 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 3.5 mg/kg, 4 mg/kg, 4.5 mg/kg, 5 mg/kg, 5.5 mg/kg, 6 mg/kg, 6.5 mg/kg, 7 mg/kg, 7.5 mg/kg, 8 mg/kg, 8.5 mg/kg, 9 mg/kg, 9.5 mg/kg, 10 mg/kg, 1 1 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, or more. It is well within the skill of a skilled artisan to determine the appropriate dose to administer to a subject.

[0059] The effectiveness of the provided methods can be monitored or assessed by any method known in the art. For example, microbial growth assays such as those described in the Examples below can be used to determine the effect of exposing microorganism to the compound. In examples where a subject is being treated by administration of auranofin, biological samples (e.g. blood, plasma, serum, sputum, saliva, urine, stool, vaginal secretions, bile, lymph, and cerebrospinal fluids, and swabs of a body surface such as skin or mucosa) can be obtained before, during and/or after administration of the compound and the presence of microorganisms in the sample can be assessed using a growth assay to determine the effect of treatment. In other examples, the presence of a microorganism in a sample taken before, during and/or after administration of the compound to a subject can be assessed using immunoassays that detect one or more microbial antigens, and such assays are well known in the art. In some embodiments, the monitoring or assessment of the methods of the present disclosure can be used to alter one or more parameters, such as duration of treatment, dose, or route of administration.

[0060] Those skilled in the art will appreciate that the aspects and embodiments described herein are susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

[0061] The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the present application. Further, the reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

[0062] The present disclosure is further described by reference to the following non- limiting examples.

EXAMPLES

Example L Effect of auranofin on the growth of S. aureus Growth Assay

[0063] A growth assay was used to determine whether auranofin could inhibit the growth of S. aureus NCTC 8325, S. aureus clinical isolates 04-229-2455, 04-228-1825, 04-227-3567 and 02-228-3611, and hospital and community acquired MRS A strains IMVS67 (nmMRSA D), RBH98 (QLD), PAH58 (SWP), MW2 (USA400), CHI 6 (UK EMRSA-15) RPAH18 (Aus-2) and USA300. [0064] Auranofin (Sigma Aidrich) was formulated as a stock concentration at 2mg/mL in DM SO, before being further serially diluted in DM SO, S. aureus strains were grown overnight in Cation-Adjusted Mueller-Hinton Broth (CaMHB) and diluted to approximately 5xl 0 'cfu/mL {i.e. 1 /100) before 150 μΐ, was added to each well of a flat bottomed 96-well plate. Three microliters of the diluted auranofin was added to the well s in duplicate. Control s included a serial dilution of lincomycin in ethanol (to assess plate to plate variation), a positive control with bacteria alone in CaMHB with 2% DMSO, and a negative (no bacteria) control with 150 μΐ CaMHB containing 2% DMSO. Plates were incubated in a shaking incubator at 37 °C for 22-24 hours (h) and absorbance was measured at a wavelength of 595 nm using a Synergy HT Bio-Tek plate reader. The growth of S. aureus was then assessed as a percentage of the positive control (bacteria alone) and the ICso values determined.

[0065] Auranofin efficiently inhibited growth of the laboratory strain of S. aureus {S. aureus NCTC 8325), exhibiting an IC?o of 0.5 _, ug/mL or 740 nM (Figure 1 A). This inhibitory effect of auranofin was also observed on S. aureus clinical isolates, with a similar ICso of 0.4-0.5 _, ug/mL (Figure IB).

Time -kill assay

[0066] The effect of auranofin on the growth of S. aureus was also assessed using a time kill assay. Exponentially growing S.aureus NCTC 8325 were diluted to approximately 5 x 10 ⁶ cfu/mL in tryptic soy broth (TSB) and auranofin, rifampicin or both auranofin and rifampicin were added. Auranofin was added to cultures at a concentration of 5 ^ig/mL, 1 _, ug/mL or 0.5 _i ug/mL. Rifampicin was added to cultures at a concentration of 1 _, ug/mL or 0.2 μg mL, while cultures with auranofin and rifampicin had 0.5 μ^ηιΕ auranofin and 0.2 /ιηΕ rifampicin. Cultures were sampled at various time points and colony forming units determined.

[0067] It was observed that the growth inhibitory effect of auranofin was both concentration- and time-dependent (Figure 2).

TUP - 1 cells

[0068] THP-1, a human monocytic cell line was infected with S. aureus (strain SH 1000-GFP) at a multiplicity of infection of 3 for 1.5. hours. Cel ls were washed 3 times and auranofin (l ug/ml in 0.5% DMSO) or 0.5% DMSO was added. Cells were observed under a fluorescent microscope at various time points post infection (Figure 3). Auranofin inhibited intracellular and extracellular S. aureus growth over a period of 24h. Infected cells treated with DMSO showed accumulation of intracellular and extracellular bacteria and cell death occurred around 7h (as judged by cell morphology).

Example 2. Effect of auranofin on 5. aureus persister cells

[0069] To determine whether S. aureus persister ceils are also susceptible to treatment with auranofin, a persister cell (or SCV) isolate hemB mutant of NCTC 8325-4 was used (Von Eiff et al., (1997) J Bacterid 179:4706-4712). This persister cell variant displays varying resistance to erythromycin and the aminoglycosides gentamicin and kanamycin.

[0070] Growth assays were performed essentially as described above with the exception that bacteria were grown in TSB, Disc assays were also performed by plating bacteria on TSB agar. Discs impregnated with 4 g auranofin, 100 μg gentamicin, or a combination of the two, were placed on top of the agar. The plates were incubated overnight at 37 °C and any zone of bacterial inhibition was observed.

[0071] Consistent with previous reports, the NCTC 8325-4 persister ceils proved to be less susceptible to gentamicin than the parental non- persister cell strain (Figure 4 A). In contrast, growth of the persister cells was inhibited by auranofin at similar concentrations (approximately I ,ug/mL) as the parental strain (Figure 4B). As shown in Figure 5, the partial inhibition of persister ceil growth observed in the presence of gentamicin (as indicated by outer ring) was reduced by the addition of auranofin.

Example 3. Intracellular activity of auranofin

[0072] The intracellular activity of auranofin was investigated using a tissue culture system in which the replication of intracellular and extracellular S. aureus NCTC8325 was assessed in the presence of auranofin, alone or in combination with other antibiotics. A549 cells were grown in RPMI 1640 medium supplemented with 10% FBS and 2mM L-giutamine. Ceils were infected with a multiplicity of infection (moi) of approximately 30 for 1.5-2h. Cells were washed and fresh medium was added containing either l ^ig/mL auranofm, 0.2^ig/mL rifampicin, both auranofin and rifampicin, or 100 units/mL penicillin and 100 }ig/mL streptomycin. Cells were harvested at various time-points. Supernatants were removed and the cfu determined. The cells were washed three times with PBS and lysed with 500 jiL of water before the intracellular cfu was determined. For later time points (longer than 24h), media was replenished with fresh media containing the appropriate compounds eve ' 24h.

[0073] As shown in Figures 6 and 7, auranofin is able to effectively penetrate cells, even in the presence of a high serum concentration (10%), and inhibit the replication of intracellular (Figures 6B and 7B and 7C) and extracellular (Figures 6 A and 7 A) bacteria in a tissue culture system at concentrations as little as 1 ^ig/mL. This effect lasted at least three days (Figure 7C). The inhibition of intracellular and extracellular S. aureus replication was enhanced using a combination of 0.2 μg/mL rifampicin and I .ug/mL auranofin (Figure 7).

Example 4. Inability of S. aureus to develop resistance to auranofin

[0074] To determine the frequency at which S. aureus generates resistance to auranofin, three separate assays were performed: a disc-diffusion assay; a resistance- frequency assay and a time-kill assay.

Disc assay

[0075] Bacteria were plated on TSB agar. 3M discs (impregnated with 4 ^ig auranofin,

4, Lig rifampicin or DMSO alone) were placed on top of the agar. The plates were incubated overnight at 37 °C and any zone of bacterial inhibition was measured. Any bacterial colonies growing within this zone of inhibition were picked and subsequently analysed for potential resistance against the appropriate compound/antibiotic using a growth as described above. No growth was observed the in auranofin zone of inhibition, however mutants were isolated from the rifampicin (Rif) zone of inhibition. These

5. aureus Rif mutants were then tested in a growth assay and their rifampicin resistance confirmed. [0076] It was observed that while both auranofm and rifampicin produced zones of inhibition around S. aureus NCTC 8325 and the various clinical isolates (including

MRS A strains IMVS67 (nmMRSA D), RBH98 (QLD), PAH58 (SWP), MW2 (USA400), CH16 (UK EMRSA-15) RPAH18 (Aus-2) and USA300), resistant bacterial colonies were only present in the rifampicin zone of inhibition (data not shown). Thus, neither S. aureus NCTC 8325 nor any of the clinical isolates were observed to develop resistance to auranofm. Conversely, colonies of nearly ail isolates were observed in the rifampicin zones of inhibition in accordance with previously published data suggesting that the mutation frequency of rifampicin is approximately 10 ^"8 (O'Neill et al. J. Antimicrob. Chemother. (2001) 47 (5): 647-650.)

Resistance frequency assay

[0077] Two resistance frequency assays were set up according to Stokes et al. (J Biol Chem (2005) 280: 39709-39715). Briefly, S. aureus was streaked out on TSB agar plates containing l , 1 .5 ^χ , 2x and 4x the minimum inhibitor}' concentration of auranofm (^g/mL). Any potential mutants were picked and assessed for resistance. The potentially resistant bacteria were cultured overnight in CaMHB broth containing serial dilutions of auranofm, rifampicin or lincomycin and assessed for their resistance profile by measuring the growth of the bacteria in a growth assay and comparing it to the control. "Wild-type" bacteria that had not originally been grown in the agar plates and which were not resistant were also included in the growth assay. "Rif resistant cells" were picked from the rifampicin zone of inhibition and potential "Au resistant" mutants were picked from the edge of the auranofm zone of inhibition and tested. It was observed that none of the potentially auranofm-resistant bacteria picked from agar plates were actually resistant, as no growth was observed in broth containing auranofm (Figure 8A&B), Conversely, the potentially rifampicin-resistant bacteria picked from agar plates were fully resistant, as these bacterial cultures grew to the same level as the controls. The wild-type (i.e. sensitive) bacteria showed no growth.

Time-kill assay

[0078] The time kill assay described above in Example 1 was observed to assess the development of auranofm- or rifampicin-resistant bacteria over time. As can be seen in Figure 2, no resistant bacteria were observed in the presence of 0.5 u u ml ., 1 fig/mL, or 5 _, ug/mL auranofm, or in the presence of a combination of 0.5 μ§/ηΊΐ. auranofm and 0.2 μg/'mL rifampicin, as indicated by the lack of bacterial growth over time. By comparison, in the presence of 0.2 μg/mL and 1 μg/mL rifampicin, bacterial growth starts to increase after about 10 hours, indicating the development of rifampicin resistance.

Susceptibility of rifampicin-resistant mutants to auranofin

[0079] The susceptibility of the rifampicin-resistant mutants to auranofm was assessed using the disc assay method. Briefly, cultures containing the confirmed rifampicin- resistant mutants were heavily streaked out on TSB agar before discs impregnated with 4 μg auranofin, 4 μg rifampicin, auranofin/rifampicin (4 ug each) or DMSO alone were placed on top of the agar. The plates were incubated overnight at 37 °C and any zone of bacterial inhibition was noted. Zones of inhibition formed around discs containing auranofin or auranofin/rifampicin but not around discs containing rifampicin or DMSO, indicating that the rifampicin-resistant mutants remained susceptible to auranofin and resistant to rifampicin (data not shown).

Conclusion

[0080] None of the S. aureus strains (either the laboratory strain or the clinical isolates) developed resistance to auranofm. Conversely, mutants with resistance to rifampicin routinely developed. Further, rifampicin-resistant mutants remained susceptible to auranofin.

Example 5. Activity of auranofin is linked with impaired redox-pathway activity and enhanced oxidative stress

[0081] Auranofin has been suggested to inhibit thioredoxin reductase in mammalian cells and recently in bacteria (Harbut et al. (2015), PNAS, 1 12: 14; 4453-4458). The thioredoxin (Trx) pathway protects bacterial cells from oxidative stress and this pathway is essential in many (but not ail) Gram-positive bacteria and some Gram- negative bacteria,

[0082] Most Gram negative bacteria possess a second pathway to deal with oxidative stress, the glutathione (GSH) and glutathione reductase pathway (GR). GR is encoded by the gor gene. GshA encodes a γ-Glutamate-cysteine ligase which catalyses the first of two steps in the pathway for the biosynthesis of GSH, GshB, a glutathione synthetase catalyses the final step in the pathway for the biosynthesis of glutathione.

[0083] To determine whether auranofin acts on the antioxidant pathway mutants from the Keio library, containing single-knockout mutants for all non-essential genes of E.coli K12 were tested in a growth assay.

[0084] Auranofin stock solution (20mg/ml) in dimethyl sulfoxide (DMSO) was serially diluted in DMSO and each dilution added in duplicate to a 96-well plate to a final DMSO concentration of 2% (v/v). An overnight culture of E.coli mutants and wild type were diluted 1 : 100 in Luria Bertani (LB) broth and 150ul of this sample was added to each well of the 96-well plates. Control wells included an 'untreated' control with bacteria in the presence of 2% DMSO and a negative sample (containing 150μ1 growth media in the presence of 2% DMSO). Plates were incubated at 370C for 22-24h and bacterial growth assessed by absorbance at a wavelength of 595nm. The minimum inhibitory concentration (MIC) was defined as the lowest concentration of compound that inhibited growth compared to the no-treatment control.

[0085] Mutants lacking gsha and gshb were more sensitive to auranofin compared to the wild-type strain (see Figure 9). By inhibiting the GSH pathway, the Trx pathway has to deal with oxidative stress. Therefore if this pathway is inhibited by auranofin, the bacteria cannot deal with the oxidative stress. This may partially also explain why Gram positive bacteria lacking the GSH pathway such as S. aureus are more susceptible to auranofin compared to Gram negative bacteria. However it is likely that this is not the only mechanism and it is interesting that the gshb mutant was not as sensitive to auranofin as the gsha mutant. Similarly, depletion of gor had little effect