AU708962B2 - Methods for microbial regulation - Google Patents

Description

WO 96/29392 PCT/AU96/00167 1 Methods for Microbial Regulation Technical Field The present invention relates to methods for regulating processes of microorganisms. In particular, methods and culture media including furanones for inhibiting homoserine lactone (HSL) and/or acylated homoserine lactone (AHL) regulated processes in microorganisms.

Background Art Microorganisms, including bacteria, fungi and algae, have a profound effect upon the activities of humans and animals. Considerable efforts have been made to find compounds and methods for the control of such micro organisms. There is a need to continually find new compounds and methods for this purpose is due, in part at least, to the capacity of such microorganisms to rapidly mutate to circumvent the compounds and methods already developed.

One set of fundamental regulator agents which are widespread in bacteria, including human pathogens, are known as homoserine lactones (HSL) or acylated homoserine lactones (AHL). The AHL regulatory systems in bacteria are two component regulatory systems which regulate intercellular activity in response to environmental conditions and extracellular signal molecules. This system was first discovered in the bioluminescent marine bacteria Vibrio harvevi and V. fischeri where it is used to control expression of bioluminescence. In principle, the system is comprised of two proteins LuxR and LuxI. The LuxI enzyme is encoded by a luxl gene and produces a related family of signal molecules known as the homoserine lactones. These signal molecules bind to the LuxR regulator which is then activated and serves both as a positive regulator for the structural genes which encode the enzymes responsible for bioluminescence, and as a positive regulator for the luxI gene itself. The entire system is amplified via a process of auto induction. Additional molecules serve as regulators of the LuxR-LuxI system.

While initially discovered for bioluminescent bacteria, this regulatory system has now been found in numerous other microorganisms.

and is involved in a wide variety of bacterial activities (Swift et al. 1994.

Fuqua et al. 1994). These activities include, but are not restricted to.

exoenzyme production in the plant pathogen Erwinia carotovora and in Pseudomonas aeruginosa, the causative agent of cystic fibrosis, and Ti WO 96/29392 PCT/AU96/00167 2 plasmid transfer from Agrobacterium tumefaciens to plants. In all instances.

acylated homoserine lactone, or homoserine lactone-like compounds are the regulatory auto inducers.

Since these regulatory systems are widespread among bacteria, and because they control processes leading to bacterial invasion of host organisms, it is likely that other organisms will have evolved defence mechanisms against these systems. So far none have been found. The present inventors have now developed methods of inhibiting microbial processes using compounds that are mimics of the homoserine lactone family of regulatory compounds.

Disclosure of the Invention Accordingly, in a first aspect the present invention consists in a method of inhibiting an homoserine lactone (HSL) and/or acylated homoserine lactone (AHL) regulated process in a microorganism comprising exposing the microorganism to a furanone compound so as to inhibit the process.

In a preferred form, the furanone compound is derived from the alga Delisea pulchra or chemical modifications or derivatives thereof. More preferably the furanone compound is selected from the group consisting of the compounds of Formula 1 and Formula 2 and even more preferably, the furanone compound is selected from the group consisting of the compounds of Figure 3.

The furanone compounds are typically used at a concentration greater than 10 ng/ml. Preferably the furanone compounds are used at concentration from 100 ng to 1 mg/ml and more preferably used at a concentration from 1 pg to 100 pg/ml.

The HSL and AHL regulated processes that can be inhibited by the present invention include motility, swarming, swimming, exoenzyme production, indigo formation, adhesion, attachment and luminescence. It will be appreciated by persons skilled in the art, however, that other AHL and/or HSL regulated processes could also inhibited by the methods of the present invention.

In order to inhibit the motility or swarming of microorganisms when cultured in vitro, the furanones used in the present invention can be included in culture media so that when the motile microorganisms are grown in or on the media their motility or swarming processes are inhibited. This WO 96/29392 PCT/AU96/00167 3 has particular utility when mixed cultures of microorganisms are cultured on semi-solid agar culture plates where there is a problem of one motile microorganism swarming over the plate and preventing the isolation or selection of other non-motile microorganisms present in the mixture. The incorporation of one or more of the furanones in a culture medium would inhibit the motility or swarming processes regulated by HSL and/or AHL of any microorganisms cultured thereon.

The method of the present invention is applicable for inhibiting processes in microbial pahtogens of plants, animals and humans in vivo.

Typical plant pathogens include Serratia liquiaciens and Erwinia carotovora, animal pathogens include Vibrio harveyi and Vibrio anguillarum and human pathogens include Pseudomonas aeurginosa and Proteus mirabilis.

It will be appreciated that the methods of the present invention can be carried out on microorganims by treating them directly with furanones as well as indirectly by, for example, giving or applying furanones to plants, animals or humans so as to inhibit HSL and/or AHL regulated processes of the microorganisms of interest in or on the plants, animals or humans. The effect of the furanones will be the reduction or inhibition of the regulated process of interest, irrespective of specific action on the HSL and/or AHL pathway itself.

In a second aspect, the present invention consists in a microbial culture medium for inhibiting an homoserine lactone (HSL) and/or acylated homoserine lactone (AHL) regulated process in a microorganism, the medium comprising growth and/or maintenance ingredients and a furanone compound.

In a preferred form, the furanone compound is derived from the alga Delisea pulchra or chemical modifications or derivatives thereof. More preferably the furanone compound is selected from the group consisting of the compounds of Formula 1 and Formula 2 and even more preferably, the furanone compound is selected from the group consisting of the compounds of Figure 3.

The furanone compound is typically included in the medium to give a final concentration in use of greater than 10 ng/ml. Preferably the furanone compound has a final concentration from 100 ng to 1 mg/ml and more preferably the furanone compound has a final concentration from 1 pg to 100 utg/ml.

As used herein the term 'a compound of Formula 1' means a compound being a furanone of the structure shown in Fig. 1, wherein R 1 is a hydrogen atom, a hydroxyl, ester or an ether group and wherein R 2 and Rg are each a hydrogen atom or a halogen atom.

As used herein the term 'a compound of Formula 2' means a compound being a furanone of the structure shown in Fig. 2 wherein R1, R2, R3 and R4 are each a hydrogen, halogen, methyl, alkyl, hydroxyl, ether or ester group.

The compounds used in the present invention consist of a series of structurally related furanones. The term "furanone compound" as used herein means an a-furanone where the carbonyl is attached to the 2 position of the furan ring (the oxygen atom occupying the 1-position in the heterocyclic ring) and such furan ring contains at least one additional degree of unsaturation. Some of these compounds (Fig. 1) have been previously described in the literature, in part by the present inventors (Kazlauskas et al., 1977; de Nys et al., 1992; de Nys et al., 1993), where their molecular structures have been elucidated and characterised by nuclear magnetic spectroscopy, mass spectrometer, ultra-violet and infra-red spectroscopy and optical rotation (ocD). The compounds all share a basic carbon skeleton consisting of a furanone moiety with an alkyl side chain at the 3-position of the basic structure. The substitution of hydrogen, hydroxy and acetoxy groups at the R 1 position, and hydrogen, bromine, chlorine and iodine at the

R

2

R

3 position is preferred and results in a large series of structurally related metabolites (Fig. The combination of the most feasible substitutions at these positions results in 30 possible structures, of which 13 have been characterised. In addition, the present inventors have now tested several synthetic analogues of these compounds (Fig. 2 and which, while sharing the basic furanone skeleton, differ in the length of their alkyl side chains.

The present invention is intended to cover inter alia, the use of all of the compounds in Fig. 1, as well as the extension of these structures shown in 30 Figs 2 and 3.

The furanone compounds are close structural analogues of the AHL family of regulators (the structure of homoserine lactone and related regulatory molecules are described in Swift et al. 1994, and Fuqua et al.

1994), and it now seems that these compounds are in fact mimics of the homoserine lactone family of regulatory compounds. Thus the furanones are Nj's likely to be broadly useful as competitive inhibitors of these regulatory systems in a wide variety of applications.

In order that the present invention may be more clearly understood examples thereof are described with reference to the following drawings:- Brief Description of the Drawings Figure 1 shows the structure of furanones from Delisea pulchra, formula 1; Figure 2 shows the structure of furanones from Delisea pulchra, formula 2; Figure 3 shows the structure of furanones tested (compounds 1-5, 8, 10 and 12-16 in assays; Figure 4 shows inhibition of AHL regulated swarming in Serratia liquifaciens by furanones; Figure 5 shows effect of furanone compound 4 on swarming LB10 with agar, swimming LB10 with 0.3% agar, and growth of Proteus mirabilus at 37 0 C; and Figure 6 shows results of the effects of crude extracts of Delisea when coated on plastic against six strains of bacteria in the laboratory and (b) attatchment of bacteria in the field.

Modes for Carrying out the Invention

METHODS

S: 20 For the tests described below, Delisea pulchra metabolites (furanone compounds) were extracted as follows: The alga was collected from Cape Banks, NSW, Australia and frozen on collection. The tissue was subsequently freeze dried, extracted with dichloromethane, and the crude extract reduced in vacuo. Purified metabolites were isolated by vacuum 25 liquid chromatography followed by high performance liquid chromatography (de Nys et al., 1993). The structural elucidation of purified metabolites was carried ut using a combination of nuclear magnetic resonance spectroscopy and mass-spectrometry techniques. Previously characterised Delisea metabolites and synthetic analogues set out in Fig. 3 were used in biological testing.

The furanone compounds and their analogous were tested against microorganisms as outlined below. All tests (bioassays) were done in the laboratory under controlled conditions.

Inhibition of homoserine lactone mediated systems in bacteria: swarming, exoenzyme, indigo production, and bioluminescence 1 i7 cv All bacteria were precultured in liquid LB medium or on LB plates at room temperature.

Serratia liquifaciens (swarming and exoprotease) assay Serratia liquifaciens was grown in liquid LB medium at room temperature to an optical density of 0.3 at 450nm and stable inoculated onto LB agar agar) plates. Plates were incubated at 30 0 C and observed regularly for swarming behaviour. The effect on swarming of furanone compounds was tested by adding (after sterilisation and cooling of the agar) compounds 1-13 (Fig. in the concentration range of 5-100 g/ml, to the plates.

For assays of production of exoproteases by Serratia liquifaciens the bacteria were cultured as above and inoculated in liquid LB medium. The effects of furanone compounds were tested by adding compounds 3 and 4 (Fig. 3) at 100 pg/ml. Aliquots were withdrawn from the cultures, centrifuged and sterile filtered. Sixty microlitres of supernatant were added to wells in skim milk agar plates skim milk in M9 medium). Plates were incubated at room temperature and observed for clearing zones around the wells.

wl The effect on growth of Serratia liquifaciens by furanone compounds 20 was also measured in liquid LB medium by comparing growth of cultures without the addition of compounds versus growth of cultures with added furanone compounds (100 ug/ml of compounds 1-13 (Fig. 3) in separate cultures). Growth was measured as optical density at 450nm and followed over a 48hr period.

Chromobacterium violencia (indigo) assay A mutant of Chromobacterium violencia negative for the HSL auto inducer was used in the assay. The strain was pour plated in LB medium at high density and wells were punched after the media had set. Sterile, S* filtered supernatants from various HSL producing strains were added to the wells. Plates were incubated at 30°C and observed for the formation of purple zones (due to indigo production) around the wells. The effects of furanone compounds 1 and 2 (Fig. 3) were assessed by running this assay on plates of LB medium containing furanone compounds at a concentration of 100/pg/ml. The effects of the compounds on growth of Chromobacterium were assessed as above.

WO 96/29392 PCT/AU96/00167 7 Pseudomonas aeruginosa (elastase) assay Inoculants from precultures were streaked onto Peptone-Tryptic Soy Broth agar containing 0.3% elastin. Plates were incubated at 37 0 C and observed for hydrolysis of the elastin. The effect of furanone compounds was assess by adding compounds 1-4 (100pg/ml) to the plates and comparing zones of hydrolysis in plates with and without the compounds.

Erwinia carotovora (exoenzyme) assay Inoculants from precultured E. carotovora were added to slices of potatoes. Degradation (softening and browning) of the potato was compared on slices with and without furanone compounds (3 and 4).

Vibriofisheri and V. harveyi (bioluminescence) assays Initial assays for both strains of Vibrio were streaked onto LB agar plates (with 2% NaC1) with and without furanone compounds 1-4 at 100 Ag/ml. Bioluminescence of the plates was visually assessed after 24 hours of incubation at room temperature. In subsequent assays, both strains were grown in liquid culture in the presence of furanones. Bioluminescence was assayed via a luminometer. The effects of furanones on growth of both strains were assayed as above.

Inhibition of surface motility and adhesion As well as specifically affecting well defined AHL mediated bacterial characteristics, furanones also inhibit other bacterial properties, in particular surface motility and adhesion. Like their interference with other AHL systems furanones interfere with or inhibit surface motility and swarming without necessarily having general growth inhibitory or microbiocidal effects. Proteus mirabilis and several characterised (but not yet taxonomically identified) marine isolates were used in these tests.

Proteus mirabilis and marine isolates (swarming assay) Methods followed those described for Serratia liquifaciens (above). Inhibition of growth of these bacteria by furanones was assessed as above.

Marine isolates (adhesion assays) Six marine bacterial strains from submerged surfaces on the coast near Sydney were isolated and cultured as above in LB medium (with 2% NaCI). Attachment assays were done in two ways. Firstly, furanones were coated onto plastic dishes at several concentrations, and the dishes attached to frames. Each frame was placed in media which contained WO 96/29392 PCT/AU96/00167 8 7 /ml of one of the 6 bacterial isolates. After 2 hours, the frames were, removed, rinsed thoroughly, stained with DAPI, and the number of attached bacteria on each dish via epifluorescence microscopy.

Secondly, inhibition of attachment was tested by preincubating the strains with furanones, so that the cells could take up the compounds. Three pre-incubation periods were used; 1/2, 3 and 10 hours.

At the end of the pre-incubation period, the cells were washed, placed into experimental containers at 10 7 /ml, and plastic dishes (uncoated) placed into the containers. After 2 hours the frames with the dishes were removed.

rinsed, and the number of attached cells counted as above.

Inhibition of growth of bacteria and fungi by compounds of Formula 2 In addition to the tests described above, the effects of selected compounds of the general formula 2 on growth of bacteria, one yeast (Candida albicans) and one fungus (Helminthosporium sp.) were assessed.

Bacteria were grown in liquid media in the presence of different concentrations of furanones and growth measured via changes in optical density Fungal assays were done by inoculating agar plates with spores. These plates contained different concentrations of furanone compounds in wells within the plates. Zones of inhibition around these wells were measured after 48 hours.

RESULTS OF BIOASSAYS Effects on AHL mediated processes The effects of furanones on AHL dependent phenotypes. and growth, for the AHL assay strains number of bacterial species are shown in Table 1. In all instances some of the furanones affected AHL dependent phenotypes at concentrations that did not inhibit growth. The most complete set of results was obtained for Serratia liquifaciens, a facultative plant pathogen. None of the furanones tested inhibited growth or swimming behaviour of this organism. All compounds tested, however, inhibited swarming, a AHL dependent behaviour, to varying degrees (Fig 4a, 4b and Table This demonstrates the presence of a mechanism that interferes with bacterial cell to cell communication and thereby limits bacterial surface colonisation without affecting growth or survival of the bacteria. In an analogous fashion, compounds 3 and 4 inhibit production of exoproteases by Serratia exoproteases at concentrations where growth is unaffected (none of the compounds tested inhibited growth of Serratia liquefaciens at 100 gg/ml).

WO 96/29392 PCT/AU96/00167 9 Indigo formation in Chromobacterium violencia, an HSL regulated system, is inhibited by compounds 1 and 2 (Table These two compounds have no effect on the growth of this bacterium. The indigo negative Chromobacterium violencia mutant did not produce the pigment in response to the addition of compounds 1 and 2 the compounds do not stimulate indigo production). As for Serratia, the compounds appear to specifically interfere with an AHL mediated process, rather than having gross effects inhibition of growth) on the organism. In both cases, the results suggest competitive inhibition of HSLs by the compounds of the invention. Growth of Chromobacterium was inhibited by 100 ug/ml of compound 4 but unaffected by the same concentration of compounds 1-3.

The compounds also affect AHL mediated processes in two important pathogens, the human pathogen Pseudomonas aeruginosa (cystic fibrosis) and the plant pathogen Erwinia carotovora (soft rot disease).

Production of elastase, which is an AHL mediated virulence factor for P.

aeruginosa, is stimulated by compounds 1 and 2, but inhibited by compounds 3 and 4. There is no detectable effect of compounds 2 and 4 on growth. The observation that structurally very similar compounds (1 and 2 versus 3 and 4) can have opposite effects on elastase production again indicates a very specific effect of the compounds on this HSL mediated system.

For Erwinia carotovora, compound 4 decreases exoenzyme production without inhibiting growth. Compound 3 has no effect on either growth or exoenzyme formation, again supportive of the specific effects of these metabolites.

Furanones inhibit AHL regulated bioluminescence in both Vibrio fischeri and V. haiveyi without inhibiting growth of either strain (Table 2).

1 Table 1. Effect of fu-ranone compounds on bacterial species with acylated homnoserine lactone phenotypes (Jrginusin Sarratia liquefaci iens Chromobcicterium IVi brioc fish eri Vibrici harveyi

&A

C

W

1

C

q4

M

u1

X

m

C

m- 0n Sigulificanco plant pathogen marine marine svmbiont svimbiont I ISL, phen)Iotype Swarig ex 1 ez es ind(igo formation luminescence luminescence acrvl-l ISL typ~e HB-IISL a.o. HB-HISL Effect of Ino effect no effect no effect no effect Fiirarioie 2 no effect no effect no effect no effect compounds 1-4 onl 3 no effect slowed no effect no effect bacterial rowth 4 no effect inhibit no effect no effect- Effect of 1 2 inhibit inhibit ni inhibit c()11)(llrlS1-4 oil IISL, phienotype inhibit inhibit 'It inhibit I. f

I

inhibit inhibit 3 inhibit decrease! inhibit inhibit inhibit In ii inIi inhibit dlecrease/ inhibit inhibit inIiil~it J I I Growth and swimming is Comni ets not inhibited by loopg/il of either compound.

100 pg/VmI of 3 and 4 decreases thle exoprotease activity of tile wild type and eliminates the exopro tease activity of Ihe 1Inli1aiit *Growth inhibited by 4 and slowed by 3 (each 100 .ig/ml *Alcmpounds inhibitory at 100 pig/mI. 4 most inhibitory V.

fish er more sensitive than V. harveyi No visible effect on luminescence of lower concentrations (based on visual inspection of agar plates 1113: N-f 3-livdroxyhiitanyl); Off: N-(3-oxhexanvl); 00: N-(3-oxvoc-vtanioyl); ODD: N-(3-oxododecanyl) nt not tested

'I

Table 1. (continued) Effect of FI'llanotie 2 conipi (11(1 1-4 onl 3 bacterialI growth 4 Pseudononas aeruginosal jErwinia carotovora Effect of FUranone fl0lliI]Oll11(I 1-4 oil I ISL, phienotype Comiimenlt s 1 2 3 4

I

opportuniistic liflail pathoge aicvstic fibrosis elastase, Exotoxin A

ODID-HSL

lit nt no effect 110 effect stimulated lit stimulated nt (delay/ lit inhibited delay/ lit Growthi onl plates 1uiiaffected( by 100 1g/ml of either compound Elastase production is also highly inedia dlepenidant no ft nt no effect inhibit plant (potato) pathogen fexoerizvlnes OIlHISL nt noeft no effect WO 96/29392 PCTAIJ96OO 167 12 Table 2. Minimum inhibitory concentrations of Delisea pulchra crude extract and furanone compounds inhibitory to four fouling relevant phenotypes: swimming, attachment, growth and swarming in strains isolated from D.

pulchr-a and rock surfaces.

Concentration of pulchra crudle extract (DC) or furanlone compound inhibiting four phIenlotypes Strain Compound Swimming Attachment Growth(2) Swarming R12 DC nid(1) 0.01 lid lid D11 >50 lid >50 D)2 >50 nid >50 1)3 50 lid >50 1)4 25 lid 25 R86 DC l 0.01 ndli DI1 50 lid >50 1)2 >50 l >50 D3 >50 li(1 >50 D4 25 11(1 25 R130 DJC 11(1 0.01 id lid Di >50 lid >50 D)2 >50 l >50 1)3 >50 11(1 25 D)4 25 lid 25 V21 D)C lid 1.0 lid nl D)1 25 11(1 25 1)2 25 l 25 1D3 25 11(1 5 1)4 5 li(1 5 V54 D)C 1l(1 1.0 nd lid D)1 5 11(1 >50 1)2 5 li(1 >50 1)3 5 l1(1 25 1)4 5 li(1 25 D)C l 1.0 l mmd D)1 5 11( >50 D)2 5 li(1 >50 1D3 5 lmil 5 14 5 l1(1 25 not dolle in liquid niedin WO 96/29392 PCT/AU96/00167 13 Inhibition of surface motility and adhesion Both crude extract of Delisea, and compound 4, inhibited swarming by the opportunistic human pathogen Proteus mirabilis.

Compound 4 did not affect growth or swimming at concentrations that were inhibitory to swarming (Fig Swarming of all 6 marine strains was also inhibited by furanones at concentrations that did not inhibit growth (Table 2).

When the crude non-polar extract of Delisea was coated onto artificial surfaces in laboratory assays it inhibited adhesion by all six marine isolates tested. Inhibition of some strains occurred at concentrations as low as 10 ng/cm 2 5 of 6 strains were inhibited by 1 ug/cm 2 (Fig 6A). Results of field assays of bacterial adhesion were consistent with these results. Crude Delisea extract coated onto surfaces at 1 ug/cm 2 decreased bacterial adhesion by >90% in the field (Fig 6B).

Pre-incubation of bacteria with crude non-polar extract of Delisea, followed by washing of the cells, also significantly inhibited subsequent adhesion of the bacteria to (uncoated) inanimate surfaces. This result is particularly telling with regard to the results for inhibition of AHL regulatory systems described above. This is because the pre-incubation experiments demonstrate that the furanones are not simply inhibiting adhesion by changing surface characteristics. Rather they are taken up by the cells, with the result that adhesion is then inhibited, and indicating that a regulatory system has been affected resulting in a loss of production of adhesin[s]. Further evidence that the regulatory system affected is AHL driven comes from the observation (data not shown) that when cells are preincubated with AHLs, adhesion increases. Although many adhesion assays were done with crude extract of Delisea. it will be appreciated by one skilled in the art that the effects are due to the furanones which are contained within the extract.

Inhibition of fungal growth by compounds of Formula 2 All compounds inhibited either Candida albicans or Helminthosporium sp. for at least one of the levels tested (Table The compounds were most effective against Helminthosporium, with activities in some cases comparable to that of the commercial fungicide Amphotericin B.

At the highest levels tested, growth of Helminthosporium was completely inhibited by 4 of the furanones: unlike the effects of Amphotericin B. It is WO 96/29392 PCT/AU96/00167 14 also emphasised that these assays underestimate the effects of these compounds relative to the commercial product, since diffusion of these compounds through agar is much less than that of the water soluble amphotericin B.

These results clearly demonstrate that natural furanones and synthetic analogues: Specifically interfere with bacterial properties that are regulated by homoserine lactone or acylated homoserine lactone regulatory systems. Specific properties known to be regulated by AHLs shown to be affected here include swarming, exoenzyme production, bioluminescence, and pigment production.

Adhesion, which is stimulated by the addition of AHLs and is also likely to be AHL regulated, was also inhibited by the furanones tested. Similarly, swarming of Proteus mirabilis and a number marine isolates, was also inhibited by the furanones. It should be noted that other AHL regulated processes or properties not tested here are also likely to be inhibited by furanones including virulence in the fish pathogen Vibrio anguillarum, plasmid transfer in Agrobacterium tumefaciens.

(ii) Some of the compounds (formula 2) tested also inhibit growth and or kill both bacteria and fungi.

WO 96/29392 WO 9629392PCT/AU96/00167 Table 3 Inhibition of fungal growth by furanones. Data are width (mil) of inhibition zone.

Candida albicans Amount Added (jig) Compound Control Ethanol Aniphotericin B Compound 1 Compound 2 Compound 3 Compound 5 Compound 8 Comipound 13 1.14 1-2 2-3 2-3 3-8 0 1 no inhibition no inhibition 6-10 5-7 0 0 0 0 1-2 0 1 0 0 0 0 0 Helminihospoium sp Amount Added (pig) 100) 10 1 0.1 Compound Contr101 E.htlaiol Aniphiotericin 13 Compoinid 1 Coiiipouind 2 Coinpouind 3 Comnpouind 5 Conipommnd 8 Conmpotnd 13 7-10 20) 20 6-20 20 20 0 no inhibition no inhibition 6-8 2-4 6-9 9-13 2 0 0 WO 96/29392 PCT/AU96/00167 16 Thus these compounds, taken as a whole, can interfere with AHL driven systems in microorganisms as well as inhibit growth of some strains. The effects are highly specific and are dependent on concentration of the furanone compounds, on the strain tested and on the structure of the furanones and on the given AHL mediated system. Furanones can be used to specifically inhibit HSL systems without inhibiting growth, as well as being used to inhibit growth. Because of the widespread occurrence of HSL mediated functions in bacteria, furanones in this context may be useful in many applications, including but not restricted to biomedical, agricultural, and antifouling applications It is noted that some previous work on the activity of some of these compounds as biocidal agents has been done, in particular the results reported by Reichelt and Borowitzka (1984). Their results show that several of the furanones of Formula 1 inhibited growth of bacteria, however, there is no indication or teaching that furanones can effect specific regulatory systems. The present inventors have found that furanones strongly inhibit specific properties (exoenzyme production, swarming, etc.) of these bacteria and these compounds can be used in a variety of in vitro and in vivo systems to effect such properties.

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 aspects as illustrative and not restrictive.

WO 96/29392 PCT/AU96/00167 17 References de Nys, Coll, Bowden, B.F. (1992). Delisea pulchra (cf. fimbriata) revisited. The structural determination of two new metabolites from the red alga Deliseapulchra. Aus. J. Chem. 45: 1625-1632.

de Nys, Wright, Konig, Sticher, 0. (1993). New halogenated furanones from the marine alga Delisea pulchra (cf. fimbriata).

Tetrahedron 49: 11213-11220.

Fletcher, R.L. (1989). A bioassay technique using the marine fouling green ala Enteromorpha. Int. Biodeterioration 25: 407-422.

Fuqua, Winans, Greenberg, E.P. (1991) Quorum sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators. J. Bacteriology 176: 269-275.

Jefford, Jaggi, Boukouvalas, J. (1988). J. Chem, Soc., Chem. Comm. 1595.

Jefford, Jaggi, Sledeski, Boukouvalas, J. (1989) Stud. Nat.

Prod. Chem., 3: 157.

Kazlauskas, Murphy, Quinn, Wells, R.J. (1977). A new class of halogenated lactones from the red alga Delisea fimbriata (Bonnemaisoniaceae). Tet. Lett. 1: 37-40.

Reichelt, Borowitzka, M.A. (1984) Antimicrobial activity from marine algae; results of a large scale screening programme. Hydrobiologia 116/117:158-168.

Swift, Bainton, N.J. Winson, M.K. (1994). Gram-negative bacterial communication by N-acyl homoserine lactones: a universal language? Trends in Microbiology 2: 193-198.

Claims (19)

1. A method of inhibiting an homoserine lactone (HSL) and/or acylated homoserine lactone (A-IL) regulated process in a microorganism, the method comprising exposing the microorganism to a furanone compound as hereinbefore defined capable of inhibiting an HSL and/or AHL regulated process in a microorganism so as to inhibit the HSL and/or AHL regulated process in the microorganism.

2. The method according to claim 1 wherein the furanone compound is derived from the alga Delisea pulchra or chemical modifications or derivatives thereof.

3. The method according to claim 1 wherein the furanone compound is selected from the group consisting of the compounds of Formula 1 and Formula 2 as hereinbefore defined.

4. The method according to claim 3 wherein the furanone compound is selected from the group consisting of the compounds of Figure 3. The method according to any one of claims 1 to 4 wherein the regulated process is selected from the group consisting of motility, swarming, swimming, exoenzyme production, indigo formation, luminescence, adhesion and attachment. 20 6. The method according to any one of claims 1 to 5 wherein the furanone compound is used at a concentration greater than 10 ng/ml.

7. The method according to claim 6 wherein the furanone compound is used at a concentration from 100 ng to 1 mg/ml.

8. The method according to claim 7 wherein the furanone compound is 25 used at a concentration from 1 tg to 100 [tg/ml.

9. The method according to claim 5 wherein the regulated process is L1L mo tility or swarming aILnd the furanone compound is incorporatedU in a microbial culture medium at a concentration greater than 10 ng/ml so as to substantially inhibit motility or swarming of the microorganism.

10. The method according to claim 9 wherein the furanone compound is incorporated in the culture medium at a concentration from 1 |tg to 100 iLg/ml.

11. The method according to claim 5 wherein the regulated process is adhesion or attachment and the furanone compound is applied to a surface at a concentration greater than 1 ng/cm so as to substantially inhibit adhesion or attatchment of the microorganism to the surface. 7" <N C i

12. The method according to claim 11 wherein the furanone compound is applied to the surface at a concentration from 10 ng to 10 pg/cm 2

13. The method according to any one of claims 1 to 12 wherein the microorganism is a pathogen of plant, crustacean, fish, animal or human.

14. The method according to claim 13 wherein the plant pathogen is selected from the group consisting of Serratia liquiaciens and Erwinia carotovora. The method according to claim 13 wherein the crustacean pathogen is Vibrio harveyi.

16. The method according to claim 13 wherein the fish pathogen is Vibrio aniguillarum.

17. The method according to claim 13 wherein the human pathogen is selected from the group consisting of Pseudomonas aeurginosa and Proteus mirabilis.

18. A microbial culture medium for inhibiting an homoserine lactone (HSL) and/or acylated homoserine lactone (AHL) regulated process in a microorganism, the medium comprising growth and/or maintenance ingredients and a furanone compound as hereinbefore defined capable of S: inhibiting an HSL and/or AHL regulated process in a microorganism. 20 19. The medium according to claim 18 wherein the furanone compound is derived from the alga Delisea pulchra or chemical modifications or derivatives thereof. The medium according to claim 18 wherein the furanone compound is selected from the group consisting of the compounds of Formula 1 and 25 Formula 2 as hereinbefore defined.

21. The medium according to claim 20 wherein the furanone compound is selected from the group consisting of the compounds of Figure 3.

22. The medium according to any one of claims 18 to 21 wherein the furanone compound is included in the medium to give a final concentration in use of greater than 10 ng/ml.

23. The medium according to claim 22 wherein the furanone compound has a final concentration from 100 ng to 1 nig/ml. i 1 ^c"y

24. The medium according to claim 23 wherein the furanone compound has a final concentration from 1 tg to 100 tg/ml. Dated this twenty-second day of February 1999 UNISEARCH LIMITED Patent Attorneys for the Applicant: F B RICE CO 9 @0* ow 9* 9 SS@ 9 0 9S 0 9000 S S S. S 0055 S SO 00 9 0* 00 S OS 0 S9 9@ 9 0S* 9* 0 9 S. U