Compositions for preventing and treating infection

Abstract

Composition including an artificial sweetener (e.g. ace-K, saccharin, sucralose, cyclamate, sugar alcohol (e.g. xylitol, mannitol, sorbitol, erythritol, maltitol, lactitol)) or a derivative thereof in

Description

The present invention relates to compositions for treating and / or preventing infection. 5 Infectious diseases are a leading cause of deaths world-wide, accounting for 25% of all deaths annually. This number would be significantly greater if it were not for antibiotics. The discovery of penicillin over 80 years ago and its subsequent uptake by healthcare 10 systems around the world revolutionised the treatment of bacterial infections. It marked the beginning of a golden age in antibiotic discovery with new classes of antibiotics being routinely discovered and saving millions of lives globally particularly in areas of the developing world. 15 However, since the beginning of the 1990s the rate of discovery has slowed to a near standstill. This lack of discovery has been compounded by the rapid emergence and spread of bacterial pathogens that exhibit resistance to multiple antibiotic treatments, including first line antibiotic 20 treatments. This has led to an antibiotic resistance crisis with deaths attributed to antimicrobial resistance reaching 4.95 million in 2019 (Murray eta!., 2022), and a predicted cumulative global cost of $100 trillion by 2050 (HM Government (2019)). 25 A 2018 report from the World Health Organisation placed Acinetobacter baumannii and Pseudomonas aeruginosa at the top of a global priority list of bacteria in urgent need of novel therapeutic intervention strategies. Research into antibiotic discovery is now a matter of global priority in order to maintain sustainable access to effective treatments 06 02 25 for bacterial infections. The rise of antibiotic resistance is closely linked to their indiscriminate use particularly in the developing world, where many antibiotics can be acquired without the need for a prescription or clinical advice. The urgent need to identify new compounds with 5 antibiotic properties has prompted scientists to explore new environments and approaches to identify potential therapeutics. The present invention seeks to provide compositions for treating and / or preventing infection. 10 According to an aspect of the present invention, there is provided a composition including ace-K in an amount sufficient to inhibit bacterial growth and / or virulence for use in a method of treating and / or preventing bacterial skin infection by a pathogen, wherein the pathogen 15 is Pseudomonas aeruginosa, Acinetobacter baumannii, and / or Stenotrophomonas maltophilia, and wherein the composition is formulated for application to a patient's skin. The virulence mechanism may be biofilm formation and / or bacterial 20 motility for example. The composition may be for use in a method of treating and / or preventing infection of a wound. 25 The composition may be formulated for application to a wound. The composition may be in the form of a liquid, cream, ointment, gel or hydrogel. 06 02 25 According to another aspect of the present invention, there may be provided a wound dressing including a composition as specified above. Embodiments of the present invention are now described, by way of 5 example only, with reference to the accompanying drawings, in which: Figure 1 schematically illustrates direct application to skin of embodiments of antibacterial compositions; Figure 2 shows that an ace-K wash of a P. aeruginosa colony biofilm can significantly reduce viable cell recovery; 10 Figure 3 shows that an ace-K wash of an A. baumannii colony biofilm can significantly reduce viable cell recovery; Figure 4 shows that an ace-K augmented wound dressing can significantly reduce viable cell recovery in P. aeruginosa colony biofilms; Figure 5 shows that an ace-K augmented wound dressing can 15 significantly reduce viable cell recovery in A. baumannii colony biofilms; Figures 6 and 7 show that an ace-K augmented wound dressing can significantly reduce viable cell recovery in A. baumannii infected burn wound or laceration in an porcine skin explant model; Figure 8 schematically illustrates oral administration of embodiments of 20 antibacterial compositions; Figure 9 schematically illustrates administration by inhalation of embodiments of antibacterial compositions; Figure 10 schematically illustrates intravenous administration of embodiments of antibacterial compositions; 25 Figures 11 and 12 show P. aeruginosa growth in the presence of saccharin; Figures 13 and 14 show P. aeruginosa growth in the presence of xylitol; Figures 15 and 16 show P. aeruginosa growth in the presence of ace-K; 06 02 25 Figures 17 and 18 show P. aeruginosa growth in the presence of sorbitol; Figures 19 and 20 show P. aeruginosa growth in the presence of maltitol; 5 Figures 21 and 22 show P. aeruginosa growth in the presence of cyclamate; Figures 23 and 24 show P. aeruginosa growth in the presence of sucralose; Figures 25 and 26 show inhibition of growth of >4. baumannii in the 10 presence of D-mannitol; Figures 27 and 28 show inhibition of growth of A. baumannii in the presence of erythritol; Figures 29 and 30 show inhibition of growth of A. baumannii in the presence of sodium cyclamate; 15 Figures 31 and 32 show inhibition of growth of A. baumannii in the presence of maltitol; Figure 33 and 34 show inhibition of growth of A. baumannii in the presence of lactitol; Figure 35 and 36 show inhibition of growth of A. baumannii in the 20 presence of xylitol; Figure 37 and 38 show inhibition of growth of A. baumannii in the presence of saccharin; Figure 39 and 40 show inhibition of growth of A. baumannii in the presence of sucralose; 25 Figure 41 and 42 show inhibition of growth of A. baumannii in the presence of ace-K; Figure 43 shows growth of P. aeruginosa in different concentrations of sir'd—* Cl L* C l\ f 06 02 25 Figure 44 shows growth of A. baumannii in different concentrations of ace-K; Figure 45 shows the ability of P. aeruginosa to form biofilm after 19 hour exposure to sweeteners in MHB medium; 5 Figure 46 shows the ability of A. baumannii to form biofilm after 19 hour exposure to sweeteners in MHB medium; Figures 47 and 48 show the minimum biofilm inhibition concentrations for P. aeruginosa and A. baumannii respectively; Figure 49 shows the key words overrepresented in a gene set 10 enrichment analysis; Figure 50 shows the impact of ace-K on bacterial motility; Figure 51 shows the impact of ace-K on natural transformation; Figure 52 shows that cation supplementation can mitigate the growth inhibition effect of ace-K; 15 Figure 53 shows antibiotic sensitivity of P. aeruginosa in the presence of ace-K; Figures 54 and 55 show antibiotic sensitivity of A. baumannii in the presence of ace-K; and Figure 56 shows antibiotic sensitivity of A. baumannii in the presence of 20 cyclamate and saccharin. The global increase in obesity due to excessive sugar consumption has propelled the discovery and inclusion of many artificial sweeteners into diets. These artificial sweeteners are FDA approved and deemed safe to 25 consume at relatively high concentrations. There are many known artificial sweeteners. They are chemically diverse, though some (such as the sugar alcohols) are chemically related. Ace-K is 200 times sweeter than sucrose. It is the potassium salt of 6-methyl-l,2,3-oxathiazine-4(3H)-one 2,2-dioxide: 5 Xylitol is a sugar alcohol having a similar sweetness to sucrose: 06 02 25 10 Mannitol is also a sugar alcohol, and is about 50% as sweet as sucrose: Erythritol is also a sugar alcohol, and is 60-70% as sweet as sucrose: Maltitol is also a sugar alcohol, having 75-90% the sweetness of sucrose: 06 02 25 Lactitol is also a sugar alcohol, and has 30-40% the sweetness of sucrose: 10 Sucralose can be 320-1000 times as sweet as sucrose. It is a derivative of sucrose containing chlorine groups: Cyclamate is 30-50 times sweeter than sucrose. It is the sodium or calcium salt of cyclamic acid. By way of example, the formula for the sodium salt is given below: Saccharin is >500 times sweeter than sucrose. It is usually used in foods in its sodium or calcium salt form: 06 02 25 20 There are many other artificial sweeteners that have been approved for use in the human diet such as sorbitol, D-tagatose, aspartameacesulfame salt, l',4,6'-trichlorogalactosucrose, glycyrrhizin, neotame, aspartame, advantame, salt of aspartame acesulfame, thaumatin and hydrogenated starch hydrolysates. Recent research has been exploring the effect that artificial sweeteners have on healthy bacteria in the gut but the findings are controversial. 06 02 25 Some studies demonstrate that the growth of gut bacteria is induced in the presence of sweeteners, while others present the opposite. WO 2014 / 082050 discloses use of sweeteners as an excipient in 5 antibacterial compositions containing antibacterial agents. However, there is no disclosure of the sweeteners themselves having any antibacterial effect. With respect to the published literature, as with the effect of artificial 10 sweeteners on human health, there is conflicting data available on the impact of artificial sweeteners on bacterial growth, for example Shahriar et al. (2020) show that acesulfame potassium (ace-K) promotes bacterial growth, as do Mahmud etal. (2019). Contrary to this positive growth effect, there is one study that mentions a negative impact on 15 growth of laboratory Escherichia coli strains (Wang et al. (2018)). The impact of artificial sweeteners on the gut microbiome has also been explored (Bian etal. (2017), Wang etal. (2018)). Sucralose has been shown to inhibit motility in the pathogen P. aeruginosa via quorum sensing inhibition (Markus et al. (2021)). 20 Two recently published studies have indicated that some artificial sweeteners, including ace-k, can promote the dissemination of antibiotic resistance genes through horizontal transfer either by natural transformation or conjugative gene transfer (Yu et al. (2021a); Yu etal. (2021b)). However these studies were not performed in pathogens. 25 As can be seen from the above discussion, studies to date on the effect of artificial sweeteners on bacterial growth have been somewhat inconsistent. To date, no-one has studied artificial sweeteners as 06 02 25 antibacterials, in particular in regard to having activity against known pathogenic bacteria. The present applicant investigated the effect of several common 5 artificial sweeteners on growth of a range of clinically relevant pathogens, and also studied their effect of a range of different virulence associated behaviours. Furthermore, the effect of these sweeteners on the efficacy of a range of commonly used antibiotics was investigated. 10 From its results, a range of artificial sweeteners with antimicrobial properties was identified. In particular, the artificial sweeteners displayed robust anti-bacterial activity against four of the six most commonly antibiotic resistant bacterial pathogens {Klebsiella pneumoniae, A. baumannii, P. aeruginosa, and Enterobacter species). 15 These pathogens are the major cause of nosocomial infections and can persist even after being treated with antimicrobial agents. The applicant has demonstrated that as well as inhibiting growth, ace-K in particular is capable of inhibiting a range of virulence behaviours such 20 as biofilm formation (associated with persistent infection) and motility (associated with dissemination throughout the body). Remarkably, at least cyclamate, saccharin and ace-K can also potentiate the activity of a range of clinically relevant antibiotics. The applicant also uncovered the mechanism of this activity using RNA sequencing. 25 The applicant proposes the use of artificial sweeteners to treat or prevent infection. Specifically, it has demonstrated that artificial sweeteners will inhibit bacterial growth and disrupt bacterial behaviours associated with virulence, including chronic infection phenotypes such 06 02 25 as biofilm formation. It has also been shown that these artificial sweeteners can potentiate the activity of a range of clinically relevant antibiotics. It is proposed that these compounds can be used to treat or prevent infection. It is envisaged that these compounds could be used to treat or prevent infections in several ways, for example topical application in the form of a liquid, cream, ointment, gel or wound dressing, oral administration, aerosolised or dry powder administration (for inhalation), or intravenous administration. In all applications, the artificial sweetener could be applied alone, or in combination with a prescribed antibiotic regime, to potentiate antibiotic activity. It is proposed that each of these applications could be used prophylactically to prevent infection or actively to treat infection. It is also proposed that each of these applications could be used in combination with antibiotic therapy to potentiate antimicrobial activity of the antibiotic. Given the current antibiotic crisis and its impact on the health care sector, there is an urgent need for novel antimicrobial treatments that can be deployed rapidly and, in some instances, deployed prophylactically to limit the overuse of antibiotic therapies. The applicant has identified a range of artificial sweeteners that can significantly impact the growth of a range of the most prevalent and problematic multidrug resistant pathogens. These compounds could be prescribed by a medical practitioner as a treatment or a prophylactic. However, given the favourable status that these artificial sweeteners have with global food and drug authorities it is possible that they may be developed as an over the counter treatment or supplement. 06 02 25 The antimicrobial activity of artificial sweeteners holds significant clinical potential. Particular advantages in using these artificial sweeteners as antibacterial compounds include: • Broad spectrum effect against multi drug resistant pathogens. • Much of pharmacokinetics and pharmacodynamics already known. • Favourable status with food and drug administrations. • Already part of the diet of many individuals. • Multi-impacts on the cell. • They can disrupt established biofilms, one of the leading causes of routine antibiotic failure. • Mechanism of action understood. • Can increase sensitivity of resistant pathogens to common antibiotics. • Can increase sensitivity to carbapenems (carbapenem resistant pathogens being a major threat to health). The skilled person will appreciate that the artificial sweeteners exemplified below could be used alone, or together in different combinations. They would also understand that chemically related derivatives of the exemplified compounds can also be used to achieve the same effects. Some embodiments can involve adding antibacterial artificial sweeteners to existing anti-infective or antimicrobial formulations. 06 02 25 EXAMPLES Example 1: Direct Application 5 In a first example, schematically illustrated in Figure 1, an artificial sweetener can be applied directly to an acute wound that is uninfected 10, an infected acute wound 12, or to a chronic wound 14. The artificial sweetener can be prepared in a desired solvent typically to 10 saturation (for example, 13.5 grams of ace-K in 50ml of sterile deionised water). The preparation is then filter sterilised through a 0.2 pm filter. This working stock solution can then be used in the preparation of all subsequent downstream applications (for example, a liquid, cream, ointment, gel, hydrogel or wound dressing). 15 By way of example a wound wash 18, taking the form of a prepared solution of artificial sweetener is used to flood the wound bed continuously over a defined time period. This area is then rinsed with sterile saline or water, and subsequently covered with a traditional 20 dressing or plaster. To explore the clinical potential of artificial sweeteners, a wash of a chronically infected wound was simulated, in order to test the effect of an ace-K wash on bacteria viability. P. aeruginosa and A. baumannii 25 colony biofilms, representing chronically infected wounds, were submerged in a 8.85% (w / v) ace-K solution for 1 hour before resuspension, serial dilution and enumeration. 06 02 25 This wash treatment led to a significant reduction in the number of viable bacteria within the biofilm for both P. aeruginosa biofilms (Figure 2) and A. baumannii biofilms (Figure 3). Data shown is average of three biological replicates with SD. Data analysis by students t test. * p < 5 0.05, ** p <0.01 *** p <0.001 versus the LB control. In another example, a gauze dressing 16 is soaked in a 10% (w / v) solution of the sweetener until saturated. This dressing can then be applied to an infected wound 12 for a defined period of time to promote 10 disinfection of the wound. The effect of ace-K loaded gauze dressing on viability of bacteria was tested. The impact of a wound dressing augmented with ace-K on chronic wound colonisation was studied. P. aeruginosa and 15 A. baumannii colony biofilms, representing chronically infected wounds, were covered with a surgical gauze soaked in a 8.85% ace-K solution for 1 hour before resuspension, serial dilution and enumeration. Treatment with the augmented dressing led to significant reductions in 20 bacteria numbers for both P. aeruginosa biofilms (Figure 4) and A. baumannii biofilms (Figure 5) compared to water-soaked dressing. Samples were tested in biological triplicate with technical quadruplets. Analysis was by independent t-test. * p <0.05, ** p <0.01 *** p <0.001 versus the LB control. 25 A similar impact was seen when these dressings were tested on a porcine ex vivo skin model. In this model, porcine skin was either burnt or lacerated and the wound infected with A. baumannii AB5075 and left for 3.5 hours to allow a biofilm to form. The dressing was applied for 1 06 02 25 hour and viable cells were collected after treatment. This resulted in a significant reduction in cells recovered compared to the water loaded dressing control. The results are shown in Figure 6 for the burn model (2.16 log reduction in viable cells versus the water control), and Figure 5 7 for the laceration model (0.5 log reduction in viable cells versus the water control). Samples were tested in biological triplicate with technical quadruplets. Analysis was by independent t-test * p <0.05, ** p <0.01 *** p <0.001 versus the water control. 10 In another example, the same dressing could be applied to an uninfected wound 10 to prevent wound colonisation by pathogens. In another example, the sweetener may be used to load a hydrogel that can be applied to the wound 10; 12; 14. 15 Comparative Example 2: Oral Administration The antimicrobial artificial sweeteners are commonly found in the diet, which means they could potentially be included as part of a patient's 20 diet to limit the risk of infection or to help potentiate the effects of antibiotics in patients that have had them prescribed, from either a GP or in a hospital setting. The ADI (acceptable daily intake) of ace-K is 15 mg per kg of body weight which is equivalent to about 1000 mg for a person weighing 75 kg. 25 We also propose a potential mouth wash or cream could be used at even higher concentrations (for example, >0.1%) to treat oral infections. 06 02 25 In an example, schematically illustrated in Figure 8, the artificial sweeteners are orally administered in the form of toothpaste 20 or chewing gum 22, for example to treat and / or prevent throat, mouth, gum and dental infections (for example, tonsillitis 24, ulcers 26, 5 abscesses 28 and tooth decay). Although chewing gum containing artificial sweetener is known, it has previously been used merely as a sugar substitute to provide sweetness. However, it is proposed that an artificial sweetener could be 10 included in chewing gum at a concentration at which it disables virulence phenotypes (such as biofilm formation). This may only require a low concentration such as less than 0.1%, less than 0.4%, less than 0.44%, less than 0.5%, less than 1%, or 0.1% to 1%. At higher concentrations, (for example, greater than 0.4%, greater than 15 0.44%, greater than 0.5%, or greater than 1%, bacteria in the mouth could be killed by the artificial sweetener. Therefore, in the example of chewing gum, the antibacterial effect would be directly from the activity of the artificial sweetener used at anti-virulence or antibacterial concentrations and not from the indirect effects of mechanical agitation 20 leading to bacterial removal or the indirect effect of sugar depletion in the oral microenvironment. Comparative Example 3: Administration by Aerosol or Dry Powder Inhalation 25 As schematically illustrated in Figure 9, the artificial sweeteners (for example, ace-K) could be administered to treat infections associated with lung disease 30 by inhalation of a dry powder preparation from an 06 02 25 inhaler 32 or an aerosolised sweetener in aqueous solution from a nebuliser 34. It is proposed that the effective artificial sweeteners could be aerosolised from a stock solution (10% w / v) using a nebuliser or inhaled as a dry powder to treat chronic infection and / or potentiate the effect of a co-administered antibiotic. Comparative Example 4: Intravenous Administration In situations where it is not possible to administer artificial sweeteners to a patient orally, they can be administered intravenously. This preparation can be an additive to a standard rehydration fluid drip or can be a separate solution where the artificial sweetener is the sole active component solubilised in a saline solution. Intravenous administration is schematically illustrated in Figure 10. Intravenous solutions 40 and / or oral solutions 42 of the artificial sweetener, at concentrations that inhibit bacterial growth / virulence, can be administered to a patient, for example to treat / prevent bacteraemia and / or sepsis. In some examples, the artificial sweetener solutions are provided at concentrations that are insufficient to have antimicrobial effect, but sufficient to augment the effect of a co-administered antibiotic 44. Example 5: Growth Inhibition Effect The effect on bacterial growth of a selection of artificial sweeteners was investigated. A standard nutrient medium was supplemented with 06 02 25 2.66% of each artificial sweetener. Control cultures were supplemented with an equal volume of the vehicle (dH20). Two specific opportunistic multidrug resistant clinically relevant pathogens were chosen for this assay: P. aeruginosa PA14 and A, baumannii AB5075. A. baumannii and 5 P. aeruginosa occupy positions one and two in the WHO priority pathogen list respectively. Cultures were incubated at 37°C with shaking, and growth was monitored over time. Growth of P. aeruginosa PA14 was measured in 10 the presence of 2.66% sweetener for 19 hours. As shown in Figures 11, 13, 15, 17, 19, 21, and 23, xylitol, sorbitol, sodium cyclamate, sucralose, maltitol, sodium saccharin and ace-K inhibit the growth of P. aeruginosa. The data depict the mean of three biological replicates ± SD. Figures 12, 14, 16, 18, 20, 22, and 24, show growth at 19 hours. 15 All of the sweeteners tested inhibit the bacterial growth with significant levels. The data present the mean of three biological replicates ± SD. ** p <0.01, *** p <0.001 versus the bacterial growth in control samples. 20 Growth of A, baumannii AB5075 was measured in the presence of 2.66% sweetener for 19 hours in LB medium. As shown in Figures 25, 27, 29, 31, 33, 35, 37, 39, and 41, xylitol, mannitol, erythritol, sodium cyclamate, sucralose, maltitol, lactitol monohydrate, sodium saccharin and Ace-K inhibit the growth of this pathogen. The data depict the 25 mean of three biological replicates ± SD. Figures 26, 28, 30, 32, 34, 36, 38, 40 and 42 show growth at 19 hours. All of the sweeteners tested inhibit the bacterial growth with significant levels. The data present the mean of 3 biological replicates ± SD. ** p <0.01, *** p <0.001 versus the bacterial growth in control samples. 06 02 25 Figures 11 to 42 thus demonstrate that xylitol, sodium cyclamate, sucralose, maltitol, sodium saccharin and ace-K all significantly inhibit the growth of P. aeruginosa and 4. baumannii, with the most 5 pronounced effects being seen with ace-K, sodium cyclamate, saccharin and sucralose. Furthermore, mannitol, meso-erythritol, and lactitol monohydrate had a significant impact on A. baumannii and sorbitol had a significant impact on P. aeruginosa. 10 To determine if this effect was dose-dependent a minimum inhibitory concentration assay was performed for both pathogens exposing them to increasing concentrations of ace-K ranging from 0.09% to 7.08% (w / v). 15 Bacterial growth was tested in 10 different concentrations of ace-K (0.09-7.08%). The results are shown in Figure 43 for P. aeruginosa and Figure 44 for A. baumannii. A statistically significant level of inhibition can be observed at 0.89% and onwards in both bacterial species. The data present the mean of three biological replicates ± SD. Data analysis 20 by independent one way ANOVA -with Tukey's post-hoc multiple comparison test to compare pairs. * p <0.05, ** p <0.01 *** p <0.001 versus the bacterial growth in control samples. A significant impact on growth was seen at 0.89% for both pathogens 25 and the effect increased with increasing concentration, plateauing at around 5%. Visual analysis of the wells in this assay suggested no growth above this concentration. 06 02 25 To demonstrate that the antibacterial effect of artificial sweeteners is present in a broad range of sweeteners, the growth inhibition experiment included xylitol, mannitol, meso-erythritol, sodium cyclamate, sucralose, maltitol, sorbitol, lactitol monohydrate, sodium 5 saccharin and ace-K. The data are three biological replicates (each with technical sextuplets), and the analysis of the endpoint ODeoo is by t-test. Example 6: Biofilm Inhibition and Dispersal 10 Biofilm formation is linked to 80% of hospital-associated infections and is a major factor in the routine failure of antibiotic therapy. To determine if any of the artificial sweeteners under study could inhibit pathogen biofilm formation, a biofilm assay was established using a 15 3% (w / v) preparation of sucralose and 2.66% (w / v) preparation of ace-K. These concentrations were chosen as although they impacted growth for the artificial sweeteners in both pathogens, they did not completely inhibit growth, therefore it should be possible to resolve an impact on biofilm formation. 20 All samples were exposed to the artificial sweeteners in LB medium for 19 hours before crystal violet biofilm assay was performed. The results for P. aeruginosa PA14 are shown in Figure 45 and the results for A. baumannii are shown in Figure 46. Different graph scales were used 25 for the different bacterial species due to the difference in their biofilm forming abilities. The data present the mean of three biological replicates ± SD. * p <0.05, *** p <0.001 versus the bacterial growth in control samples. 06 02 25 As can be seen from Figures 45 and 46, artificial sweeteners differently influence the formation of biofilm in both bacterial strains. Ace-K and sucralose can significantly inhibit biofilm formation by P. aeruginosa and A. baumannii. To determine the full impact of ace-K on A. baumannii and P. aeruginosa biofilm formation a minimum biofilm inhibition concentration assay was performed. A range of ace-K concentrations was used. All samples were exposed to artificial sweeteners for 19 hours before crystal violet biofilm assay was performed. The results are presented in Figures 47 (P. aeruginosa} and 48 {A. baumannii). The data present the mean of three biological replicates ± SD. Data analysis by independent one-way ANOVA -with Tukey's post-hoc multiple comparison test to compare pairs. * p <0.05, ** p <0.01 *** p <0.001 versus the bacterial growth in control samples. This assay revealed that at 0.09%, a significant impact on biofilm production could be seen in A. baumannii. This implies that ace-K has anti-virulence properties as well as antibacterial properties as the concentration is below that which impacts bacterial growth. For P. aeruginosa a concentration of 1.77 % resulted in an almost complete abolition of biofilm formation. Example 7: Impact of Ace-K on Gene Expression Given that the most pronounced effects on growth and biofilm formation were observed for A. baumannii and ace-K, RNA-seq analysis was performed to determine the influence ace-K had over gene expression 06 02 25 in A. baumannii. Cells were grown to early exponential phase (OD 0.6-0.7) in 20 ml LB supplemented with either 1.34% ace-K or the matching volume of vehicle control. Cells were spun down and washed in RNAIater to preserve mRNA. RIMA was isolated using a Qiagen 5 RNAeasy Kit with column DNAase digestion. RNA integrity was determined using a Bioanalyzer. Samples were further processed for RNA sequencing on an Illumina MiSeq with 12 million reads per sample. Quality control and adapter trimming was performed with bcl2fastq. Read mapping was performed with HISAT. Differential expression 10 analysis was performed using edgeR's exact test for differences between two groups of negative-binomial counts with an estimated dispersion value of 0.1. 464 genes were identified as being significantly differentially expressed greater than | logFC| >1 and p <.05 (Table 1). 15 Table 1 Locustag Gene logFC PValue ABUW_0020 -1.466497832 0.000187017 ABUW_0030 1.131184384 0.002567219 ABUW_0031 ppc -1.662752483 5.08E-05 ABUW_0066 hppD 2.752392366 2.46E-06 ABUW_0068 2.934595319 1.34E-05 ABUW_0069 maiA 3.066678875 1.43E-06 ABUW_0070 fahA 2.656580577 1.24E-05 ABUW_0071 aroPl 2.705121681 4.33E-05 ABUW_0072 1.649882584 2.51E-05 ABUW_0083 -1.169711141 0.000348967 ABUW_0088 -1.30717283 8.34E-05 ABUW_0104 -1.392962697 0.00140037 ABUW_0117 -1.016081429 0.005726823 ABUW_0121 -1.608118722 0.000883377 ABUW_0133 -1.590578804 0.000200506 ABUW_0143 2.019352617 7.77E-06 ABUW_0144 -1.157897762 0.02207712 ABUW_0154 1.073665908 0.000275842 06 02 25 ABUW_0160 -2.548549696 1.48E-05 ABUW_0203 gabT -1.121557601 0.00305788 ABUW_0225 -1.53286278 9.57E-05 ABUW_0248 -2.784516767 4.44E-05 ABUW_0259 1.866484379 0.002857042 ABUW_0263 -3.533749325 1.51E-05 ABUW_0280 sbp -2.180402608 3.20E-05 ABUW_0290 -2.750354273 0.000244176 ABUW_0291 comN -3.437791867 5.85E-05 ABUW_0292 comO -3.180420132 0.000111766 ABUW_0293 comL -3.553965663 fl i rr or O. / 0 0-1)3 ABUW_0294 comQ -3.182923601 □ IOC HE □ . iy t-uo ABUW_0304 -5.413509044 3.66E-07 ABUW_0306 bfrl -1.130984988 0.011409578 ABUW_0307 2.72139885 0.006597702 ABUW_0313 fimT -2.408510092 0.000368468 ABUW_0314 pilV -3.197287217 9.00E-05 ABUW_0315 pilW -2.646553666 0.00018702 ABUW_0316 pilX -2.351181112 0.000105213 ABUW_0317 pilY -2.569853292 2.03E-05 ABUW_0318 comE -2.118684292 0.000111502 ABUW_0319 comF -1.822946469 3.28E-05 ABUW_0356 2.685891775 1.51E-06 ABUW_0375 -1.01121916 0.000429532 ABUW_0377 hemF -1.134263602 0.014706827 ABUW_0386 mlaC 1.15915729 0.000422654 ABUW_0387 mlaB 1.126772992 0.000792293 ABUW_0437 -1.181318166 0.001922308 ABUW_0461 -1.366538508 0.000126973 ABUW_0469 -1.260191826 0.001185083 ABUW_0498 -1.038870197 0.002687295 ABUW_0514 -2.071185747 0.000142172 ABUW_0527 -1.188703686 4.62E-05 ABUW_0528 radC -1.053311891 0.000121621 ABUW_0534 -2.644159862 4.46E-06 ABUW_0535 -2.125038625 1.00E-05 ABUW_0547 1.198585588 3.93E-05 ABUW_0548 1.070564147 0.001867164 ABUW_O55O -1.029404612 0.00020632 ABUW_0553 -1.367693461 0.000449378 ABUW_0554 -1.250883348 0.000129916 ABUW_0571 -1.838461955 0.00210141 06 02 25 ABUW_0585 -1.311483704 0.000560258 ABUW_0600 1.204184781 0.000165549 ABUW_0603 6.286028154 5.83E-07 ABUW_0607 4.309477176 5.47E-07 ABUW_0641 -1.523170143 0.000156602 ABUW_0642 gcd -1.184166008 0.000775223 ABUW_0648 -1.261801918 0.00057305 ABUW_0677 -2.24450084 5.83E-05 ABUW_0678 pilG -1.868612819 0.000255176 ABUW_0679 pilH -1.947111824 9.77E-05 ABUW_0680 pill -2.789060235 8.03E-05 ABUW_0681 pilJ -3.199017969 4.03E-06 ABUW_0682 pilL -3.848722689 1.28E-06 ABUW_0683 -3.425360452 1.54E-06 ABUW_0684 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0.001925156 ABUW_3204 -1.774507562 0.000531221 ABUW_3286 1.675035131 3.81E-05 ABUW_3332 -1.192400584 0.016835846 ABUW_3337 -1.486084593 7.78E-06 ABUW_3342 dusA 1.211677065 0.000464639 ABUW_3343 2.522352648 4.82E-06 ABUW-3344 1.02241681 0.000230469 ABUW_3351 -2.050059891 1.90E-05 ABUW_3358 -2.169352924 1.30E-05 ABUW_3362 1.647924064 1.50E-05 06 02 25 ABUW-3363 macB 1.211669142 4.91E-05 ABUW_3364 1.279225684 5.18E-05 ABUW_3396 pta -1.051048081 0.003659447 ABUW_3403 2.085710276 0.001970168 ABUW-3424 1.247617239 0.000280309 ABUW_3425 1.601687763 7.66E-05 ABUW_3426 1.86329375 1.30E-05 ABUW_3439 -1.262171408 0.000141913 ABUW_3453 -2.680084197 3.24E-05 ABUW_3459 -1.930952177 4.23E-06 ABUW_3487 -1.069963228 0.000900451 ABUW_3488 -1.059660199 0.000271872 ABUW_3499 -1.076848539 0.000366387 ABUW_3514 -1.422807477 4.57E-05 ABUW_353O -1.188567309 0.000644493 ABUW_3549 pilB -2.443848945 0.000121919 ABUW_355O pile -2.608063399 0.000176643 ABUW_3551 pilD -1.40303889 0.001110607 ABUW_356O 1.492538144 SzIF-i) i ABUW_3561 2.448738584 0.000149119 ABUW_3562 2.380654266 1.31E-06 ABUW_3571 -2.014921895 0.003283463 ABUW_3579 -1.178798443 0.005939728 ABUW_3588 1.255206501 0.000400077 ABUW_3622 1.305929983 0.000398844 ABUW_3633 feoA 1.080397955 0.007001617 ABUW_3641 pilR -9 9^^07077^ Z. Zj□ 27 / _7Z / □ 4.32E-06 ABUW_3706 -1.090558097 0.001381673 ABUW_3719 1.106906107 0.000327954 ABUW_3722 -1.725802982 0.000238975 ABUW_3723 dprA -1.724800459 0.000210078 ABUW-3730 1.198680597 0.000232347 ABUW_3777 2.040025143 c 7) a c a c □ .ZOt-UD ABUW_3781 3.068956725 8.83E-05 ABUW_3782 mmsB 2.55114889 0.000586866 ABUW_3783 mmsAl 2.645326526 0.000153743 ABUW_3787 -1.576076275 0.000667821 ABUW_3788 dadX -1.156138247 0.001186136 ABUW_3789 dadA2 -1.237507612 0.001765981 ABUW_3797 -1.386412119 0.007701785 ABUW_3798 -2.452729969 3.12E-06 ABUW_3803 -1.015795252 0.007815163 06 02 25 ABUW_3804 -1.399747108 0.005873496 ABUW_3805 -1.259125525 0.000257829 ABUW_3811 did -3.015086242 6.09E-08 ABUW_3812 IldD -3.450472456 1.33E-07 ABUW_3813 IldR -3.254882636 9.47E-08 ABUW_3814 IldP -5.111652801 1.46E-08 ABUW_3838 nadC 1.166238698 0.000858396 ABUW_3839 2.232228138 3.63E-05 ABUW_3851 4.225812632 2.22E-06 ABUW_3874 2.210294507 0.000304541 ABUW_3875 1.413495025 0.001884202 ABUW_3878 grpE -1.077426106 0.000327062 ABUW_3879 dnaK -1.146049607 0.000789385 ABUW_3880 -1.19152474 0.000702505 ABUW_3884 -1.235183269 0.013983442 ABUW_3887 -1.058275224 0.001531793 ABUW_3892 -1.19783562 0.000176112 ABUW_3899 dsbC2 i ncQnciQ / i 1 .UbJUd / o4 0.001053195 ABUW_4021 2.021860601 0.00019345 ABUW_4031 -1.412510812 0.000223039 ABUW_4032 -1.546866393 0.003635406 ABUW_4069 1.247746584 0.004170976 ABUW_4071 1.030385032 0.000754874 ABUW_4072 1.181300821 0.000223564 ABUW_4087 1.022806857 0.006205437 ABUW_4116 -1.066065598 0.000475097 Analysis of this panel of genes revealed that almost all genes involved in pilus production and natural transformation were significantly down- 5 regulated (Table 2). Table 2 Table 2 shows differentially expressed genes associated with pilus assembly and function and natural competency. All genes were down 5 regulated compared to vehicle control. 06 02 25 Locus Tag Name Function Log2FC ABUW_0290 type IV pilus assembly protein PilM ; ABUW_0291 co mN type 4 fimbria! biogenesis protein PtfN : -3.43778 ABUW_0292 comO pilus assembly protein, PHO : ABUW 0293 co ml pilus assembly protein, PHQ : ABUW 0294 comQ fimbrial assembly protein PifQ : -3.38293 ABUW 0304 type IV pitin structural subunit 111111111111 ABUW_0306 bfrl bacterioferritin -1.13C98 ABUW_0313 fimT pilin protein FimT -2.40851 ABUW_0314 pilV type IV pilus modification protein PHV : -3.13728 ABUW_0315 pilW pilus assembly protein PilW -2.84655 ABUW_0316 piM pilus assembly protein PilX : -2.35118 ABUW_0317 pilY pilus assembly protein tip-associated adhesin PilY I -2.56985 ABUW_0318 comE pilin like competence factor ABUW 0319 co mF pilin like competence factor -1.82295 ABUW 0648 type 4 fimbrial biogenesis protein FimT -1 2618 ABUW 0677 hypothetical protein -2.2445 ABUW_0678 pitG type IV pilus response regulator receiver protein PtiC -1,86861 ABUW_0679 pUH type IV pilus response regulator protein PilH -1.94711 ABUW_0680 pit! type IV pilus signal transduction protein Pill : -2.78965 ABUW_0681 pit! type IV pilus methyl-accepting chemotaxis sensoiy tr 3.W9QB ABUW_0682 pitL type IV pilus hybrid sensor kinase / response regulatc^^^^^^ ABUW_0683 hypothetical protein : 3.42S3S ABUW_0684 coproporphyrinogen 111 oxidase 2-62005 ABUW 0685 alpha / beta hydrolase fold protein -1 42347 ABUW 2255 pitZ type 4 fimbrial biogenesis protein -1 43S58 ABUW2310 fimbrial protein -2,5-6433 ABUW_2311 pili assembly chaperone -1 71119 ABUW_2312 fimbrial biogenesis outer membrane usher protein -1.29&45 ABUW_2313 fimbrial protein -1 41119 ABUW_3031 pilT twitching mobility protein -2,31048 ABUW_3032 pitU twitching motility protein ; -2,34772 ABUW_3549 pits type IV-A pilus assembly ATPase PifB ; -244385 ABUW 3530 pitc pilin biogenesis protein -2,60866 ABUW 3551 piiD type IV pilus prepilin peptidase PilD ■1.40304 ABUW 3641 pHR type 4 fimbriae expression regulatory protein PifR -2,23398 06 02 25 This was a particularly interesting finding as in both Yu et al. studies, ace-K increased the expression of these genes albeit at lower concentrations (Yu et al. (2021a); Yu etal. (2021b)). 5 Gene set enrichment analysis within the subset of differentially expressed genes identified "3D-structure" and "cell inner membrane" as overrepresented key words (Figure 49). This suggests that genes associated with the cell membrane and 3D cell structure were overrepresented in the set of differentially expressed genes. This points 10 to ace-K having a role in altering the bacterial cell membrane. Example 8: Motility Effect Motility is a central facet of bacterial virulence and facilitates bacterial 15 dissemination to the blood stream or other sites within an infected host. The transcriptomic data suggested that ace-K could inhibit the expression of genes associated with A. baumannii twitching motility. To validate the gene expression data, we performed twitching assays at a range of different concentrations. In agreement with the gene 20 expression / transcriptomic data, a significant reduction in bacterial twitching motility to as low as 0.66% ace-K was observed. Figure 50 illustrates the results, in which the data are derived from three biological replicates. 25 These results further support the capacity of ace-K to have an antivirulence effect on bacterial pathogens. 06 02 25 Example 9: Natural Transformation Effect A key finding from the two Yu et al. studies was that ace-K could promote natural transformation and as such promote the acquisition of 5 antibiotic resistance genes (Yu et al. (2021a); Yu et a / .(2021b)). This finding is contrary to the transcriptomic data obtained by the present applicant. To investigate further, the impact of ace-K on natural transformation on the multidrug resistant strain of A. baumannii AB5075 was tested. Remarkably and in accordance with the applicant's 10 transcriptomic data and motility data, supplementation of growth media with ace-K led to a significant reduction in natural transformation. The results are shown in Figure 51. It was found that supplementation of media with 1.33% ace-K led to a significant reduction in 15 transformation efficiency, in contrast to the findings of Yu et al. Data shown is average of five biological replicates with SD. Data analysis by students t test. * p <0.05, ** p <0.01 *** p <0.001 versus the bacterial transformation in control samples. 20 Example 10: Cation Supplementation to Mitigate Growth Inhibition by Ace-K The transcriptomic data suggested that the bacterial cell membrane may be significantly altered upon exposure to ace-K. If ace-K disrupts 25 membrane permeability, it ought to be possible to mitigate this through the addition of exogenous cations. To explore this possibility the growth assays described above in Example 1 were repeated, but in media supplemented with magnesium and calcium cations. These cations are known to help maintain membrane stability. 06 02 25 A. baumannii AB5075 and P. aeruginosa clinical isolate G4R7 were grown in LB, LB including ace-K, and LB including ace-K and further supplemented with Mg2+ and Ca2+ cations. Remarkably (as can be seen in Figure 52) the addition of cations to both A. baumannii AB5075 and P. aeruginosa G4R7 partially restored the growth inhibition observed in the presence of ace-K. Given that ace-K had such a pronounced impact on the growth of P. aeruginosa and A. baumannii, and that the mechanism was through membrane disruption, it was hypothesised that it may have the same effect against other clinically relevant pathogens. To explore this, growth assays in the presence of 2.66% were conducted. Eight different bacterial species {A. baumannii AB5075, P. aeruginosa G4R7, E. coli BM16, Stenotrophomonas maltophilia, Klebsiella pneumoniae. Staphylococcus aureus, Enterococcus faecalis and Enterobacter cloacae) were grown in LB, LB including ace-K, and LB including ace-K and further supplemented with Mg2+ and Ca2+ cations. The results are shown in Figure 52. Data shown is average of three biological replicates with SD. Data analysis by students t test. * p <0.05, ** p <0.01 *** p <0.001 versus the LB control. Remarkably, this assay demonstrated that ace-K significantly inhibits the growth of Enterococcus faecalis, Enterobacter cloacae, E. coli BM16, Stenotrophomonas maltophilia, and Klebsiella pneumoniae, but not Staphylococcus aureus. The finding that both Gram-negative and Grampositive bacteria were impacted by ace-K highlights the potency of its 06 02 25 activity. S. aureus did have a minor reduction in growth in the presence of ace-K, but this was not significant. The supplementation of the media with Mg2+ and Ca2+ cations was able 5 to at least partially reverse the inhibitory effect of ace-K on P. aeruginosa, A. baumannii, E. coli BM16, Stenotrophomonas maltophilia, Klebsiella pneumoniae and Enterobacter cloacae. As membrane permeability can be mitigated through the addition of 10 exogenous cations, the hypothesis that membrane disruption is responsible for ace-K effect on growth is supported by these data. Comparative Example 11: Antibiotic Potentiation Effect 15 The disruption of the membrane by ace-K suggests that bacteria may be rendered more susceptible to antibiotic treatment in the presence of ace-K. To explore this hypothesis, the susceptibility of A. baumannii and P. aeruginosa to a panel of different antibiotics in the presence and absence of a sub-minimum inhibitory concentration of ace-K was tested. 20 P. aeruginosa was grown in the presence of ace-K for 19 hours, and exposed to commonly used antibiotics (gentamicin, piperacillin / tazobactam, and polymyxin B). Figure 53 shows that this resulted in an increased susceptibility to gentamicin, 25 piperacillin / tazobactam and polymyxin B. The data present the mean of three biological replicates ± SD. * p <0.05 versus the bacterial growth in control plates. 06 02 25 In the case of 4. baumannii, ace-K potentiates the activity of gentamicin, polymyxin B, doripenem, imipenem and meropenem (Figures 54 and 55). 5 A. baumannii AB5075 is known to be resistant to carbapenems. It was grown on agar plates without ace-K, or with 2.2% or 2.4% ace-K. Discs impregnated with polymyxin B, gentamicin, meropenem, imipenem and doripenem were added to the plates. The results are shown graphically in Figure 55 where exposure to ace-K led to significant increase in the 10 size of the zone of clearance for each antibiotic. Minimum of three biological replicates for all except doripenem which only has one. Data analysis by students t test. * p <0.05, ** p <0.01 *** p <0.001 versus the control. 15 Figure 54 shows a visual representation of zones of clearance of A. baumannii AB5075 around an E strip for doripenem, imipenem and meropenem with 0%, 2.2% or 2.4% Ace-K. The E strip is impregnated with a concentration gradient of the antibiotic with highest concentration at the top and lowest at the bottom. It can be seen that 20 in the presence of ace-K a zone of inhibition is visible around the E strip for each of the antibiotics compared to the control which only has water added to the agar. The data presented is a representative image of three biological replicates ± SD. 25 The presence of ace-K significantly increases the sensitivity of a multidrug resistant strain of A, baumannii AB5075 to aminoglycosides (such as gentamicin), polymyxins (such as polymyxin B), and betalactams, including carbapenems, (such as doripenem, meropenem, imipenem, and piperacillin). 06 02 25 The antibiotic potentiate effect was also seen for cyclamate and saccharin, where plates loaded with 2.66% of each sweetener and paper discs impregnated with antibiotics (imipenem and doripenem) were placed on the plate and the zone of clearance measured after 24 5 hours of growth. This was then compared to the control plate (Figure 56). The applicant has demonstrated that artificial sweeteners, such as ace-K, can inhibit the growth of a range of the most clinically relevant 10 pathogens. They can also inhibit a range of different virulence associated behaviours in those pathogens. Remarkably some of these sweeteners have been shown to augment the efficacy of a range of antibiotics in clinical use. The therapeutic application of these sweeteners could have a major impact on tackling infection, in 15 particular multidrug resistant infections. The skilled person will appreciate that derivatives of the exemplified artificial sweeteners, and compounds related thereto could equally be useful in the compositions and methods described herein. 20 Given that these sweeteners, in particular ace-K, saccharin and sucralose, are already present in the diet at relatively high concentrations, the potential to repurpose these compounds as therapeutic agents is promising. The applicant has not identified any previous disclosure relating to the use of, in particular, ace-K or 25 sucralose as antibacterial compounds. All optional and preferred features and modifications of the described embodiments and dependent claims are usable in all aspects of the invention taught herein. Furthermore, the individual features of the 06 02 25 dependent claims, as well as all optional and preferred features and modifications of the described embodiments are combinable and interchangeable with one another. 5 References Bian et al. (2017) PLoS ONE 12, e0178426 HM Government (2019) "Tackling antimicrobial resistance 2019-2024: The UK's five-year national action plan" Dept, of Health and Social Care, 10 policy paper. Mahmud et al. (2019) J. Mol. Microbiol. Biotechnol. 29, 43-56 Markus etal. (2021) Int. J. Mol. Sci. 22, 9863 15 Murray etal. (2022) Lancet 399, 629-55 Shahriar etal. (2020) MetaboL Open 8, 100072 20 Wang et al. (2018) PLoS ONE 13, e0199080 Yu etal. (2021a) ISMEJ. 15, 2117-30 Yu etal. (2021b) ISMEJ. 16, 543-54 25 06 02 25

Claims

1. A composition including ace-K in an amount sufficient to inhibit bacterial growth and / or virulence for use in a method of treating and / or 5 preventing bacterial skin infection by a pathogen, wherein the pathogenis Pseudomonas aeruginosa, Acinetobacter baumannii, and / or Stenotrophomonas maltophilia, and wherein the composition is formulated for application to a patient's skin.10 2. A composition for use as claimed in claim 1, wherein the pathogenis a multidrug resistant pathogen.

3. A composition for use as claimed in claim 1 or 2, wherein the infection is caused by Pseudomonas aeruginosa or Acinetobacter 15 baumannii.

4. A composition as claimed in any preceding claim, wherein the infection is caused by Pseudomonas aeruginosa.20 5. A composition as claimed in any preceding claim, wherein theinfection is caused by Acinetobacter baumannii.

6. A composition for use as claimed in any preceding claim, wherein the method of treating and / or preventing the bacterial skin infection 25 includes inhibiting biofilm formation.

7. A composition for use as claimed in any preceding claim, wherein the method of treating and / or preventing the bacterial skin infection includes inhibiting bacterial motility.

8. A composition for use as claimed in any preceding claim, wherein the composition is for use in a method of treating and / or preventing infection of a wound.

9. A composition for use as claimed in any preceding claim, wherein the composition is formulated for application to a wound.1010. A composition as claimed in any preceding claim, in the form of a liquid, cream, ointment, gel or hydrogel.

11. A wound dressing including a composition as claimed in claim 9 or10.

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