AU759182B2 - Inhibition of gram positive bacteria - Google Patents

Description

WO 99/53915 PCT/AU99/00284 1 Inhibition of Gram positive bacteria Technical Field The present invention relates to the use of furanone compounds as antibacterial agents, particularly as antibacterial agents for Gram positive bacteria.

Background Art It is known that a variety of furanone compounds possessing antifungal and antimicrobial properties can be isolated from red marine algae Delisea fimbriata. Delisea elegans and Delisea pulchra (Reichelt and Borowitzka (1984) Hydrobiologia 116: 158-168). When first isolated, it was thought that these compounds may be suitable as antimicrobial agents for use in animals including humans. Unfortunately, it was found that most if not all of these naturally occurring compounds were toxic to animal cells at the concentrations required to inhibit microorganisms and therefore unsuitable for many veterinary and medical applications.

Gram positive bacteria are a major problem in hospitals, on skin, in the dental area. for heart transplants, catheters, and other biomedical implants.

Unfortunately, not all antimicrobial agents are active against Gram positive bacteria. Gram positive bacteria are also present in domestic areas like bathrooms, toilets and kitchens and can also cause a disease hazard for these sources. Accordingly, there is a need for more agents that are suitable to inhibit or kill these types of microorganisms in many varied situations including domestic, veterinary and medical applications.

The present inventors have now made the surprising finding that new synthetically produced furanone compounds have inhibitory activity against Gram positive bacteria without having corresponding deleterious activity againsl animal cells previously reported to be the case with naturally occurring furanones.

Disclosure of Invention In a first aspect, the present invention consists in a method of inhibiting the growth of a Gram positive bacterium, the method comprising treating the bacterium with an effective amount of one or more furanones having the Formula as set out in Figure 1, wherein the effective amount of the one or more furanones does not substantially adversely effect the survival of an animal cell when exposed to the one or more furanones.

WO 99/53915 PCT/AU99/00284 2 In a preferred embodiment of the first aspect of the present invention, the furanone has the formula 2, 24, 25, 26, 27, 30, 33, 34, 45, mixtures and/or racemic mixtures thereof as shown in Figure 2.

Preferably. the furanone is selected from is compound 2, 30, 45 or a mixture of compounds 33/34. More preferably, the furanone is a mixture of compounds 33/34 or compound 2.

Although compounds 33/34 have been found to be particularly suitable as antimicrobial agents, it will be appreciated, however, that other furanones may have similar or even greater activity against Gram positive bacteria. The finding by the present inventors that furanones other than the known naturally occurring compounds (for example compound 4) have activity against Gram positive bacteria without being substantially toxic to animal cells may lead to the production and use of other furanone compounds.

Accordingly. it will be appreciated that the present invention covers other such compounds.

The present inventors have found that a concentration of a furanone of about 500 ng/ml was effective against a Gram positive bacterium. Higher concentrations were also effective but importantly were not toxic to animal cells. II will be appreciated that even lower concentrations may also be active against certain Gram positive bacteria and the present invention is not limited to the concentrations as tested and described herein.

The concentrations of furanones found to be active against Gram positive bacteria are not inhibitory against the Gram negative bacteria presently tested by the present inventors. The activity of the furanones are therefore surprisingly selective in their inhibitory action for Gram positive bacteria.

In a second aspect, the present invention consists in the use as an inhibitory agent against Gram positive bacteria of an effective amount of a furanone having the Formula as set out in Figure 1, wherein the effective amount of the furanone do not substantially adversely effect the survival of an animal cell when exposed to the furanone.

In a preferred embodiment of the second aspect of the present invention, the furanone has the formula 2, 24, 25, 26, 27, 30, 33, 34, mixtures and/or racemic mixtures thereof as shown in Figure 2.

WO 99/53915 PCT/AU99/00284 3 Preferably. the furanone is selected from is compound 2, 30, 45 or a mixture of compounds 33/34. More preferably, the furanone is a mixture of compounds 33/34 or compound 2.

The active furanones can be used as antibacterial agents for direct administration to mammals; as additives or preservatives for medical/surgical devices. disinfectants, soaps, shampoos, hand washes, denitrifies, household cleaning formulations, detergents for laundry and dishes; in wash and treatment solutions for topical use, instruments and devices including contact lenses, and other disinfecting and antibacterial applications The active furanones can be formulated as an antiseptic, disinfectant or antimicrobial agent. It will be appreciated that the formulations will be particularly useful in situations where it is necessary to inhibit or kill Gram positive bacteria without causing any deleterious effects on animal or mamnialian cells.

In a third aspect, the present invention consist in a method of inhibiting the growth of a Gram positive bacterium in a subject infected with the bacterium. the method comprising administering to the subject an effective amount of one or more furanones having the Formula as set out in Figure 1. wherein the effective amount of the one or more furanones do not substantially adversely effect cells of the subject.

In a preferred embodiment of the third aspect of the present invention, the furanone has the formula 2, 24, 25, 26, 27, 30, 33, 34, 45 mixtures and/or racemic mixtures thereof as shown in Figure 2.

Preferably. the furanone is selected from is compound 2, 30, 45 or a mixture of compounds 33/34. More preferably, the furanone is a mixture of compounds 33/34 or compound 2.

In a fourth aspect, the present invention consists in a pharmaceutical composition active against Gram positive bacteria, the composition including one or more furanones having the Formula as set out in Figure 1 together with one or more pharmaceutically acceptable diluents or excipients, wherein the one or more furanones do not substantially adversely effect animal cells at the inhibitory concentration used in the composition.

In a preferred embodiment of the fourth aspect of the present invention. the furanone has the formula 2, 24, 25, 26, 27, 30, 33, 34, mixtures and/or racemic mixtures thereof as shown in Figure 2.

WO 99/53915 PCT/AU99/00284 4 Preferably, the furanone is selected from is compound 2, 30, 45 or a mixture of compounds 33/34. More preferably, the furanone is a mixture of compounds 33/34 or compound 2.

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

In order that the present invention may be more clearly understood, preferred forms will be described in the following examples with reference to the accompanying drawings.

Brief Description of Drawings Figure 1 shows the general structure of furanones suitable for the present invention wherein R 1 is a hydrogen atom, a hydroxyl, ester or an ether group and wherein R 2 and R 3 are each a hydrogen atom or a halogen atom.

Figure 2 shows the structure of furanone compounds formula 2, 24, 26. 27. 30. 33. 34, 45 tested for activity against Gram positive bacteria.

Figure 3 shows the results of the effect of furanone compounds 2, 24/25 and 30 on the growth of Staphylococcus aureus 29.

Figure 4 is shows the results of the effect of furanone compounds 2, 24/25 and 30 on the growth of S. aureus 015.

Figure 5 is shows the results of the effect of furanone compounds 2, 24/25 and 30 on the growth of S. epidermidis 017.

Modes for Carrying Out the Invention METHODS AND RESULTS Furanone compounds A number of furanone compounds having structures shown in Figure 2 were tested directly on Gram positive bacteria. Synthetically produced furanones are often produced as mixtures of isomers 33/34, 26/27, etc.).

It will be appreciated, however, that variations in these structures are similar enough to be expected to have an effect, and thus the present invention includes within its scope furanones with the more general structure shown in Figure 1 which do not substantially adversely effect the survival of an animal cell when exposed to the furanone.

WO 99/53915 PCT/AU99/00284 Microbiology An initial screening of six different furanones against growth of Staphylococcus aureus and Staphylococcus epidermidis was performed in a BioRad 3550 Microplate Reader. Ten percent of an overnight culture (10 cells/nil resulting in final cell densities of 106 cells/ml was added to the growth media. Nutrient Broth, containing furanones at the concentrations and 1 [tg/ml. The bacteria were incubated at 37°C and growth was measured at 595 nm for 9 hours. Cytotoxicity in mammalian systems was measured as inhibition of the growth of mouse fibroblast cells.

The results showed several important features. First, the furanone compounds strongly inhibited growth at effective concentrations 1 and ug/ml) comparable to that of standard commercial antibiotics. Second, synthetic furanone compounds or mixtures of furanone compounds 24/25: 26/27: 33/34) were often more active than those furanone compounds which are produced naturally by the red algae Delisea pulchra, the main natural source of these fimbrolides. Third, a number of these furanone compounds were inhibitory at concentrations well below concentrations that inhibited mouse fibroblasts. That is, the furanone compounds inhibited bacteria at non-toxic (to mammals) concentrations. For example, compounds 30 and 33/34 were the most active against both Staphylococcus aureus and Staphylococciis epidermidis (Table completely inhibiting growth at ug/ml. However, cultured mouse cells were not affected by compound 33/34 (for example) until 50 ug/ml.

WO 99/53915 PCT/AU99/00284 6 Table 1. Activity of six different furanones against growth (after 9 hr) of two Grain positive bacteria and a mouse fibroblast cell line.

Staphylococcus aureus Staphylococcus epidermidis Mouse line Compound 10 pg/ml 1 ig/ml 10 ig/ml 1 pg/mg (pg/ml *33/34 0 73.0 b 0 80.0 0 56.6 62.2 72.1 *26/27 77.4 82.1 19.9 71.6 *24/25 58.2 80.1 47.7 62.2 #4 34.3 84.2 53.2 77.1 1 #2 57.9 87.9 53.2 63.7 150 Lowest concentration of furanones which significantly inhibited the growth of mouse fibroblast cell line L292. A 30% depression of growth is deemed significant growth compared to the control value synthetically produced furanones natural products The tests described above, while clearly demonstrating the efficacy of these compounds particularly the synthetic furanones against Gram positive bacteria, are unrealistic for many veterinary and medical applications in that they combine a very nutrient rich media with a very high initial inoculum density. In many clinical situations, colonising bacteria are much less dense initially, or will invade or contaminate much less nutrient rich media saline). Thus a series of tests were done with a lower density innocula, or in less nutrient rich media. These experiments used compound 33/34, the most active synthetic mix in the initial screen.

Low numbers of overnight cells (Staphylococcus aureus) were inoculated to three different media; Nutrient broth and 10% and 5% of NB each diluted in 0.9% NaC1 solution containing compound 33/34. This gave a final amount of 10 cells/ml media. The samples were incubated at 37 0 C and growth was measured at 610 nm. Three different concentrations of compound 33/34 were tested, 500 ng/ml, 1 pg/ml and 5 g/ml. Results reported are for 8 days (192 hr) and 19 days (456 hr) are shown in Table 2.

WO 99/53915 PCT/AU99/00284 7 Table 2. Growth of Staphylococcus aureus (final amount of 10 cells/ml) in various media containing compound 33/34.

Concentration of compounds 33/34 Medium Control 500 ng/ml 1 pg/ml Nutrient broth (NB) 8 h" growth after 30 hr c NB'1 45 h b c NB" 61 h b c a Time taken for control cultures to initiate growth b No growth after 192 hr c no growth after 456 hr d NB media diluted with 0.9% NaCi solution Table 2 shows clearly that in these realistic growth conditions, concentrations of 33/34, as low as 500 ng/ml, completely inhibited the growth of S. amreus. These concentrations are two orders of magnitude less than those which affect mouse cell lines. The inhibitory concentrations are also lower than those used for any current commercial antibiotic against Gram positive bacteria.

Occular Applications It has been reported that natural furanones have ability to inhibit the adhesion and swarming in a range of marine bacteria at concentrations that did not effect growth. In order to investigate whether different side chain would play any anti-bacteria roles, the effect of synthetic fuanones (at lower concentrations) on growth of different ocular bacterial strains were examined.

MATERIALS AND METHODS Bacteral strains Baclerial strains included the commonly isolated ocular microorganisms listed in Table 3.

WO 99/53915 PCT/AU99/00284 8 Table 3. Ocular gram-positive bacteria used in the study Strain Gram stain Source S. aureus 001 Gram-positive cocci Type strain S. aiureus 015 Gram-positive cocci Type strain S. aureiis 029 Gram-positive cocci CLPU S. epidermidis 017 Gram-positive cocci CLARE Corlynebacterium 003 Gram-positive rods Infiltrative keratitis Furanone compounds Compounds 2, 24/25, 26, 30, 33/34 and 45 were examined for their effects on Ilie growth of the bacterial strains. The concentrations used in the study for all the tested compounds were within the non-cytotoxic ranges.

Effect of furanone compounds on the growth of bacteria in TSB Strains were cultured in 5 ml of TSB with or without SUs in 350 C water bath with agitation (120 rpm). Growth rates were monitored by reading at time intervals of 1, 2, 3, 4, 5, 7, and 24 hours.

Eflect of furanone compounds on the growth of bacteria in artificial tear fluid (AFT) The formulation of ATF used in the study was reflected in the composition of closed-eye tears. It contained 2% of human serum.

Overnight growing bacteria were harvested and washed once with PBS.

After resuspending in ATF to OD 66 o 0.1 (108 cells/ml), 200 g 1 of bacterial cell suspension was mixed with a same volume of ATF containing furanone compounds in a Bijio bottle. The final concentration of furanone compounds was 20 Ag/ml. The cultures were then incubated at 350 C for 24h. Viable counts were quantified by plating out serial dilution of the ATF culture onto nutrient agar.

Minimum inhibition concentration of furanone compounds One hundred /l/well of TSB containing two-fold serial diluted furanone compounds (at the concentration range from 20 to 1.25 ig/ml) were distributed in 90 well of microtitre plate. Ten u1 of 10 4 cells/ml overnight bacterial culture was added in each well to get the final concentration of cell numbers of 10 cells/ml. After incubation for one or two days at 35 0

C,

bacterial growth was measured at Abs 45 0 in a microtitre reader.

WO 99/53915 PCT/AU99/00284 9

RESULTS

Effect of furanone compounds on the growth of Gram positive bacteria in

TSB

The growth curves showed in Figures 3 to 5 demonstrated that furanone compound 2 and furanone compounds 24/25 inhibited the growth of S. arens and S. epidermidis strains at the tested concentrations of 100 gg/ml and 20 pg/ml respectively; furanone compound 30 only slowed down the grow l of S. aureus and S. epidermidis strains at lower concentrations g/ml. Figure There were only less than 8% growth of S. aureus strains after 24 h incubation time in the presence of furanone compounds 2 and 24/25 (Table For Gram positive rods Coiynebacterium sp 003, furanone compound 2 inhibited approximately 86% of growth at 100 g/ml. Furanone compounds 24/25 (20 pg/ml) and 30 (10 gg/ml) only slowed down the growth of Corynebacterium sp 003 (Table as the growth reached 71 and 90% in the presence of furanone compounds 24/25 (20 ug/ml) and 30 (20 gg/ml) respectively, after 24 h incubation.

Table 4. Percentage growth of Gram positive bacteria in the presence of furanone compounds compared to the control values (after 24h).

furanone furanone furanone Strain compound 2 compounds compound 100 [g/ml 24/25 10 ~g/ml ig/ml G+ cocci: S. aureus 015 8 5 97 S. epidermidis 017 7 3 91 G+ rods: Corynebacteriuml 003 14 71 Low numbers of overnight cells (Staphylococcus aureus) were inoculated to NB diluted in PBS (buffered NaCI solution) containing furanone compounds 33/34. This gave a final amount of 25 cells/ml media. The samples were incubated static at room temperature and growth was measured bv CFU (colony forming unit). One concentration of furanone WO 99/53915 PCT/AU99/00284 compounds 33/34 was tested, 5 jg/ml. Results reported are for 2 days, 5 days and 38 days and reported in Table Table 5. Growth of Staphylococcus aureus (final number of cells/ml) in 5% NB media containing furanone compounds 33/34.

Time (days) Control Furanone compounds 33/34 (5 ug/ml) 2 10 5 cells/ml no growth 10 4 cells/ml no growth 38 10 3 cells/ml no growth The results in Table 5 shows that growth of S. aureus was clearly inhibited at concentration 5 /g/ml.

A screening of six different furanone compounds against growth of Staphylococcus aureus, Staphylococcus epidermidis. Streptococcus salivarius, Streptococcus pyogenes, Enterococcus faecalis and Micrococcus luteus was performed in a BioRad 3550 Microplate Reader. Ten percent of overnight cultures were added to the growth media. NB containing furanones at the concentration 10 /zg/ml. The bacteria were incubated at 37 0 C and growth was measured at 595 nm for 24 hours. The results are shown in Table 6.

Table 6. Activity of different furanone compounds (at concentration of ug/ml) against growth (after 24 hr) of six Gram positive bacteria. Results given as growth compared to the control value.

Furanone Compound Slaph.

(lureus Staph.

epidermidis Strep.

salivarius Strep.

pvogenes Entero.

faecalis Micro.

luteus 4 22 69 26 85 100 33/34 0 0 0 0 62 63 0 62 73 50 54 86 26/27 77 20 92 84 75 24/25 58 48 87 72 92 4 34 53 85 0 75 3 22 32 86 14 89 2 58 53 86 87 77 86 WO 99/53915 PCT/AUU99/00284 11 Table 6 shows that all the different furanone compounds tested inhibit the growth of Gram positive bacteria and that the furanones act in a species specific manner. However, compound 33/34 demonstrated a broader range of inhibitory activity compared to the other compounds and also completely preveneds growth of four Gram positive bacteria.

DISCUSSION

Prior art (Reichelt and Borowitzka (1984) Hydrobiologia 116: 158-168) showed an initial indication that naturally produced furanones can inhibit the growth of Gram positive bacteria at probably relatively high concentrations. However, the authors concluded that furanones were not useful as antibacterial agents in mammalian systems because they were toxic. The investigators based these conclusions primarily on tests with crude extracts of Delisea pulchra in which compound 4 is one of the more abundant furanones. The present inventors found compound 4 to be quite toxic against mammalian cell lines but determined that a number of other furanone compounds are necessarily toxic to mammalian cells.

Surprisingly, the present inventors have shown that the results with compound 4 are not generally representative of the effects of other furanone compounds. which are for the most part strongly inhibitory against Gram positive bacteria at non-cell line inhibitory concentrations. Moreover, synthetic furanone compounds tested were more effective than naturally occurring compounds.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims (24)

1. A method of inhibiting growth of a Gram positive bacterium, the method comprising treating the bacterium with an effective amount of one or more furanones having the following Formula: R, R4 R 2 R 3 O 0 Br wherein R, is selected from the group consisting of hydrogen, a hydroxyl 10 group, Br, F, an ester group and an ether group, R2 and R3 are individually .o selected from hydrogen or halogen and R4 is selected from hydrogen or CH3(CH2)n, where n is 0, 2 or 4, wherein the effective amount of one or more wherein Ri is selected from the group consisting of hydrogen, a hydroxyl 10 group, Br, F, an ester group and an ether group, R2 and R3 are individually selected from hydrogen or halogen and R 4 is selected from hydrogen or CH3(CH2)n, where n is 0, 2 or 4, wherein the effective amount of one or more furanones does not substantially adversely affect the survival of an animal cell when the animal cell is exposed to the one or more furanones at the effective amount. S° 2. A method of inhibiting growth of a Gram positive bacterium, the method comprising treating the bacterium with an effective amount of one or more furanones having the formula 2, 4, 24, 25, 26, 27, 30, 33, 34, 45 mixtures and/or 20 racemic mixtures thereof as shown in Figure 2, wherein the effective amount of the one or more furanones does not substantially adversely affect the survival of an animal cell when exposed to the one or more furanones.

3. The method according to claim 2 wherein the one or more furanones is a mixture of compounds 33 and 34.

4. The method according to claim 2 wherein the one or more furanones is compound 2. m:\pak\species\102750clmpak.doc 13 The method according to any one of claims 1 to 4 wherein the concentration of the one or more furanones is about 500 ng/ml.

6. Use as an inhibitory agent against Gram positive bacteria of an effective amount of one or more furanones having the following Formula R1 R4- R2 R 3 0 0 Br wherein R 1 is selected from the group consisting of hydrogen, a hydroxyl 10 group, Br, F, an ester group and an ether group, R2 and R3 are individually selected from hydrogen or halogen and R 4 is selected from hydrogen or CH 3 (CH 2 where n is 0, 2 or 4, wherein the effective amount of one or more Sfuranones does not substantially adversely affect the survival of an animal cell when the animal cell is exposed to the one or more furanones at the effective amount.

7. Use as an inhibitory agent against Gram positive bacteria of an effective amount of one or more furanones having the formula 2, 4, 24, 25, 26, 27, 30, 33, 34, mixtures and/or racemic mixtures thereof as shown in Figure 2, wherein the 20 effective amount of the one or more furanones does not substantially aversely effect the survival of an animal cell when exposed to the furanone.

8. The use according to claim 7 wherein the one or more furanones is a mixture of compounds 33 and 34.

9. The use according to claim 7 wherein the one or more furanones is compound 2. The use according to any one of claims 6 to 9 or one or more furanones as antibacterial agents for direct administration to mammals; as additives or preservatives for medical/surgical devices, disinfectants, soaps, shampoos, hand m:\pak\species\102750clmpak.doc washes, dentifrices, household cleaning formulations, detergents for laundry and dishes; in wash and treatment solutions for topical use, instruments and devices including contact lenses, and other disinfecting and antibacterial applications.

11. The use according to any one of claims 6 to 9 of one or more furanones formulated as an antiseptic, disinfectant or antimicrobial agent.

12. A method of inhibiting the growth of a Gram positive bacterium in a subject infected with the bacterium, the method comprising administering to the subject an effective amount of one or more furanones having the following Formula R SR4 /R2 R3 0 0 Br wherein Ri is selected from the group consisting of hydrogen, a hydroxyl group, Br, F, an ester group and an ether group, R 2 and R3 are individually o selected from hydrogen or halogen and R 4 is selected from hydrogen or 0 CH 3 (CH 2 where n is 0, 2 or 4, wherein the effective amount of the one or more furanones does not 2 substantially adversely affect cells of the subject. a

13. A method of inhibiting the growth of a Gram positive bacterium in a subject infected with the bacterium, the method comprising administering to the subject 0 an effective amount of one or more furanones having the formula 2, 4, 24, 26, 27, 30, 33, 34, 35, 45 mixtures and/or racemic mixtures thereof as shown in Figure 2, wherein the effective amount of the one or more furanones does not substantially adversely affect cells of the subject.

14. The method according to claim 13 wherein the one or more furanones is a mixture of compounds 33 and 34. m:\pak\species\1 02750cImpak.doc The method according to claim 13 wherein the one or more furanones is compound 2.

16. A pharmaceutical composition active against Gram positive bacteria, the composition including one or more furanones in an amount effective to inhibit growth of the bacteria having the following Formula R1 R4 R 2 R3 *2 .o *o *0 0 0 S: Br 10 wherein RI is selected from the group consisting of hydrogen, a hydroxyl group, Br, F, an ester group and an ether group, R 2 and R3 are individually selected from hydrogen or halogen and R 4 is selected from hydrogen or CH 3 (CH 2 where n is 0, 2 or 4, together with one or more pharmaceutically acceptable diluents or excipients, wherein the effective amount of the one or more furanones does not adversely affect the survival of animal cells at the effective amount.

17. A pharmaceutical composition active against Gram positive bacteria, the composition including one or more furanones having the formula, 2, 4, 24, 20 26, 27 30, 33, 34, 45 mixtures and/or racemic mixtures thereof as shown in Figure 2 in an amount effective to inhibit the growth of the bacteria together with one or more pharmaceutically acceptable diluents or excipients, wherein the effective amount of the one or more furanones does not substantially adversely affect animal cells.

18. The composition according to claim 17 wherein the one or more furanones is a mixture of compounds 33 and 34.

19. The composition according to claim 17 wherein the one or more furanones is compound 2. m:\pak\species\102750clmpak.doc Use of one or more furanones having the following Formula R, R4 R 2 R 3 O O Br wherein Ri is selected from the group consisting of hydrogen, a hydroxyl group, Br, F, an ester group and an ether group, R 2 and R3 are individually Oo selected from hydrogen or halogen and R4 is selected from hydrogen or CH 3 (CH 2 where n is 0, 2 or 4, 10 in the manufacture of a medicament for inhibiting growth of a Gram positive bacterium in a subject infected with the bacterium, wherein the one or more furanones is present in an amount effective to inhibit growth of the positive bacterium but not substantially adversely affect cells of the subject.

21. Use of one or more furanones having the formula 2, 4, 24, 25, 26, 27, 30, 33, 34, 45 mixtures and/or racemic mixtures thereof as show in in Figure 2 in the manufacture of a medicament for inhibiting a Gram positive bacterium in a subject infected with the bacterium, wherein the one or more furanones is 2present in an amount effective to inhibit growth of the bacterium, but not to substantially adversely affect cells of the subject.

22. A furanone compound of having activity against a Gram positive bacterium having the following Formula H Br H Br Ik\species\1 0 2 7

24. An antibacterial composition comprising an effective amount of compound of the following Formula and a diluent or carrier, Sees S 5 S* S.* S S S 55 S S S.* S 55* S S S wherein RI is selected from the group consisting of hydrogen, a hydroxyl group, Br, F, an ester group and an ether group, R2 and R3 are individually selected from hydrogen or halogen and R 4 is selected from hydrogen or CH 3 (CH 2 where n is 0, 2 or 4, wherein the effective amount of the one or more furanones is effective to inhibit the growth of a Gram positive bacterium but not to substantially adversely affect animal cells, and a diluent or carrier. 10

25. A composition of matter comprising an effective amount of at least one compound of the following Formula wherein Ri is selected from the group consisting of hydrogen, a hydroxyl group, Br, F, an ester group and an ether group, R2 and R3 are individually selected from hydrogen or halogen and R4 is selected from hydrogen or CH 3 (CH 2 where n is 0, 2 or 4, wherein the effective amount inhibits the growth of a Gram positive bacterium but does not substantially adversely affect the survival of animal cells, m:\pak\species\102750cimpak.doc the composition selected from the group consisting of disinfectant compositions, soaps, shampoos, hand washes, dentifrices, cleaning compositions, laundry detergents, dishwashing detergents, and tropical compositions. 23. A medical or surgical device comprising an effective amount of a compound of the following Formula R, R4 R 2 R 3 0 :*wer Br wherein R is selected from the group consisting of hydrogen, a hydroxyl 0 10 group, Br, F, an ester group and an ether group, R2 and R 3 are individually selected from hydrogen or halogen and R 4 is selected from hydrogen or CH 3 (CH 2 where n is 0, 2 or 4, wherein the effective amount inhibits the growth of a Gram positive bacterium but does not substantially adversely affect the survival of animal cells. A medical device according to claim 24 wherein the device is a contact lens.

26. A medical device according to claim 24, wherein the device is a biomedical 2 implant.

27. A medical device according to claim 24, wherein the device is a catheter.

28. A method according to any one of claims 1 to 5, and 12 to 15, wherein the Gram positive bacterium is selected from Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus salivarius, Streptococcus pyogenes, Enterococcus faecalis, Micrococcus luteus and Corynebacterium.

29. A use according to any one of claims 6 to 11, and 20 to 21, wherein the Gram positive bacterium is selected from Staphylococcus aureus, Staphylococcus m:\pak\species\102750clmpak.doc 19 epidermidis, Streptococcus salivarius, Streptococcus pyogenes, Enterococcus faecalis, Micrococcus luteus and Corynebacterium. A composition according to any one of claims 16-19 and 25 wherein the Gram positive bacterium is selected from Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus salivarius, Streptococcus pyogenes, Enterococcus faecalis, Micrococcus luteus and Corynebacterium.

31. A device according to any one of claims 23 to 27, wherein wherein the Gram positive bacterium is selected from Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus salivarius, Streptococcus pyogenes, Enterococcus faecalis, Micrococcus luteus and Corynebacterium. 1 :0 Dated this fifth day of February 2003 Unisearch Limited Patent Attorneys for the Applicant: F B RICE CO S S S o m:\pak\species\102750clmpak.doc