WO2007075595A2 - Biofilm assay - Google Patents

Abstract

The invention describes methods for measuring biofilms or biofilm formation by microorganisms using compounds that fluoresce upon contacting certain biofilms, e.g. thioflavin, styrylbenzene, and naphthalene derivates. The methods can be used to detect biofilms in vitro and in vivo, and to screen for compounds that modulate biofilms.

Description

BIOFILM ASSAY

[001] This application claims the benefit of the U.S. Provisional application No. 60/751,790, filed on December 20, 2005, which is incorporated herein by reference.

BRIEF DESCRIPTION OF THE INVENTION

[002] The invention relates to methods for measuring biofϊlms or biofilm formation by microorganisms using compounds that fluoresce upon contacting certain biofilms.

BACKGROUND

[003] Biofilms are sessile microbial communities embedded in a self-produced extracellular matrix. A number of microorganism form biofilms, including bacteria, yeasts, fungi, algae and protozoa. Biofilm formation occurs in two stages. The first stage involves cells attaching to a surface, which may be mediated, at least in part, by cell wall associated adhesins, including the microbial surface components recognizing adhesive matrix molecules. The second stage of biofilm formation includes cell multiplication and formation of a mature, multi-layered structured community. The second stage is associated with the production of extracellular factors, such as the polysaccharide intercellular adhesin (PIA) produced by the IcaADBC glycosyltransferase.

[004] Biofilms have a special clinical relevance as biofilm producing microorganisms are a leading cause of nosocomial infections. Implantation of artificial devices such as central venous catheters are a major etilogical agent of the infections. The microorganisms adhere to artificial devices as a biofilm and thrive, leading to persistent bloodstream infections and increased mortality, morbidity and risk of endocarditis. Biofilm associated microorganisms have an innate resistance to antibiotics, disinfectants and clearance by host defenses. These properties likely contribute to the persistence and recalcitrance to treatment of biofilm infections.

[005] Biofilm formation also adversely affects public health and has important implications in medicine, drinking water systems, water cooling systems, industrial fluid processing systems and food processing systems. Consequently, there is a need to develop assays that can be used to detect and identify biofilms, and which can be used to screen for compounds that are useful as modulators of biofilms.

SUMMARY OF THE INVENTION [006] In general, the invention relates to a method for measuring biofilms or biofilm formation. The method utilizes fluorescent compounds and compositions to detect biofilms.

The fluorescent compounds and compositions utilized in the method exhibit selective fluorescence by providing an increased level of fluorescent emission upon contacting and/or interacting with biofilms relative to the level of fluorescent emission produced by the compound or compositions alone.

[007] In a first aspect, the invention provides a method for measuring biofilms using thioflavins, thioflavin derivatives, styrylbenzenes, styrylbenzene derivatives or naphthalene derivatives.

[008] In a second aspect, the invention provides a method for measuring biofilms comprising the steps of providing a vessel containing a growth medium; adding a thioflavin, a thioflavin derivative, a styrylbenzene, a stytylbenzene derivative or a naphthalene derivative and measuring fluorescence.

[009] In an embodiment of the first and second aspects, the invention features a method for measuring biofilms by providing a thioflavin or a thioflavin derivative of formula I

or a salt thereof, wherein the variables X₅ Y, Ri, R₂, R₃, R₄, R₅ and R₆ are defined herein. [010] In another embodiment, the compound is a styrylbenzene or a styrylbenzene derivative of formula VII

VII or a salt thereof, wherein the variables R₂₂, R₂₃ and R₂₄ are defined herein.

[011] In yet another embodiment, the compound is a styrylbenzene or a styrylbenzene derivative of formula VIII

. VIII or a salt thereof, wherein R₂s is defined herein. [012] In a further embodiment, the compound is a naphthalene derivative of formula IX

IX or a salt thereof, wherein R₂6 and R₂₇ are defined herein.

[013] In even yet another embodiment, the method includes the step of providing a vessel, wherein the vessel is coated with a biotic coating such as serum proteins. In a further embodiment, the vessel can include a medical device or portion thereof. [014] In an even further embodiment, the assay can be used to detect and quantitate microorganisms that produce the biofilms and interact with the fluorescent compounds. The microorganisms comprise microorganisms that can synthesize polysaccharide intercellular adhesion molecules (PIA) or other polysaccharides. For instance, the microorganism includes a Staphylococcus species.

[015] In a third aspect, the invention provides a method for screening for compounds that modulate biofilms or biofilm formation.

[016] In a fourth aspect, the invention includes a kit for assaying a biofilm. [017] In a fifth aspect, the invention features a method to detect and/or measure a biofilm in vivo using thioflavins, thioflavin derivatives, styrylbenzenes, styrylbenzene derivatives or naphthalene derivatives. In one embodiment, the method includes detecting binding of the compounds to a biofilm by gamma imaging, magnetic resonance imaging and/or magnetic resonance spectroscopy. In another embodiment, putative biofilms are detected in immunocompromised patients and/or patients suspected of having an infection caused by microorganisms.

[018] In a sixth aspect, the invention provides methods for detecting and measuring biofilms in biopsies and post mortem tissue.

[019] In some embodiments, the method can be used to qualitatively detect the presence of biofilms and the microorganism that create biofilms. In other embodiments, the method can be used to quantitate the amount of biofilm and microorganisms that create biofilms by comparing amount of fluorescence from a sample containing an unknown amount of biofilm or microorganisms with a calibration chart that plots the fluorescence intensity versus amount of biofilm or microorganisms in known control samples. [020] Advantageously, the assay of this invention provides 1) selective detection of biofilms formed by specific microorganisms, 2) high sensitivity, 3) a methodology for high throughput screening; and 4) real time monitoring (in vitro and in vivo) and detection of biofilms.

BRIEF DESCRIPTION Qg THE DRAWING

[021] Figure 1 shows the emission profile of Thioflavin T in the presence and absence of S. epidermidis.

DETAILED DESCRIPTION I. Definitions

[022] To facilitate an understanding of the invention, a number of terms are defined below.

[023] As defined herein, "biofilm" is a population of microorganisms comprised of a single species or multiple species that are adhered to an abiotic or biotic surface or to each other, or at any interface. By "biofilm formation" is meant the formation, growth and modification of the microorganisms contained in the biofilm, as well as the synthesis and maintenance of the exopolysaccharide matrix of the biofilm structures.

[024] As used herein, a "microorganism," "microbial organism" or "microbe" is a microscopic, single-celled organism that may live either independently or as part of a multicellular community or colony. The major groups of microorganisms include, without limitation, archaea, bacteria, fungi, protozoa and algae.

[025] As used herein, a "raw environmental sample" is a sample of microorganisms taken from any environment. The environments include, without limitation, the mouths of animals and humans, other body cavities of animals and humans, standing water, lakes, other bodies of water, household and industrial environments and air samples.

[026] By "modulating" is meant changing, by increase, decrease or otherwise. The change may be in amount, timing, or any other parameter.

[027] By "polysaccharide intercellular adhesion molecule" or "PIA" is meant a molecule that is produced by a microorganism that is or is structurally similar to poly-β-(l→6)-N- acetylglucosamine. Sadovskaya, et al.. Infect. Imm. (2005) 3007-17. "Structurally similar to

PIA" means a molecule that has a three dimensional structure similar to PIA.

[028] By "Calgary Peg plate" is meant the plate described in U.S. Patent No. 6,596,505 or similar devices.

[029] By "surface material" is meant a material that is added to the vessel used to grow the biofilm. The surface material can be, without limitation, material used for a medical device such as a catheter, stent, valve or other device. The material can, without limitation, artificial material such as Teflon or silicone of can be derived from a natural source, such as an acellular heart valve obtained from an animal. The material can also be derived from plants or microorganisms.

[030] As used herein a "flow cell" is a device that allows for the examination of growing biofilms by microscopy. For example, U.S. Patent No. 5,641,458 discloses a flow cell. [031J By "structurally similar" is meant a structure that has a similar three-dimensional structure. The structure can be very similar with only some side chains differing, or it can be a structure that is chemically different, such as a polysaccharide and a polypeptide, but the three dimensional structure is similar enough so that small molecules can recognize both structures. [032] For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 87th Ed., (2006). Additionally, general principles of organic chemistry are described in "Organic Chemistry" 2^nd ed., Thomas Sorrell, University Science Books, Sausolito: 2006, and "March's Advanced Organic Chemistry", 6th Ed., Ed.: Smith, M.B. and March, J., John Wiley & Sons, New York: 2007, the entire contents of which are hereby incorporated by reference. [033] As used herein the term "aliphatic' encompasses the terms alkyl, alkenyl, alkynyl. [034] As used herein, an "alkyl" group refers to a saturated aliphatic hydrocarbon group containing 1 -8 (e.g., 1-6 or 1-4) carbon atoms. A lower alkyl refers to a C₁-C₄ alkyl. An alkyl group can be straight or branched. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-heptyl, or 2- ethylhexyl. An alkyl group can be optionally substituted with one or more substituents such as halo, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy, aroyl, heteroaroyl, alkoxycarbonyl, alkoxycarbonylamino, alkylcarbonyloxy, nitro, cyano, amino, acyl, sulfonyl, sulfinyl, sulfanyl, sulfoxy, urea, thiourea, sulfamoyl, sulfamide, oxo, carbamoyl, cycloalkyloxy, heterocycloalkyloxy, aryloxy, heteroaryloxy, aralkyloxy, heteroarylalkoxy, or hydroxyl. Without limitation, some examples of substituted alkyls include alkoxyalkyl, alkylaminoalkyl, amino alkyl, (heterocycloalkyl)alkyl, alkylsulfonylalkyl, alkylsulfonylaminoalkyl, alkylcarbonylaminoalkyl, alkylaminoalkyl, alkylcarbonylalkyl, alkylsulfonylaminoalkyl, hydroxyalkyl, alkoxycarbonylalkyl, alkylcarbonylaminoalkyl, alkoxycarbonylaminoalkyl, alkylcarbonylalkyl, cyanoalkyl, hydroxyalkyl, carbonylalkyl, carboxyalkyl, oxoalkyl, aralkyl, alkoxyaralkyl, (alkylsulfonylamino)alkyl, (sulfonylamino)alkyl, carbonylaminoalkyl, aminocarbonylalkyl, cycloaliphaticalkyl, cyanoalkyl, aminoalkyl, oxoalkyl, alkoxycarbonylalkyl, (alkoxycarbonylheterocycloalky^alkyl, (cycloalkyl)alklyl, (cycloalkenyl)alkyl, (heterocyclόalkyl)alkyl, or halόalkyl.

[035] As used herein a "lower alkly group" is an alklyl group containing 1-3 carbon atoms. [036] As used herein, an "alkenyl" group refers to an aliphatic carbon group that contains 2-8 (e.g., 2-6 or 2-4) carbon atoms and at least one double bond. Like an alkyl group, an alkenyl group can be straight or branched. Examples of an alkenyl group include, but are not limited to, allyl, isoprenyl, 2-butenyl, and 2-hexenyl. An alkenyl group may be optionally substituted with one or more substituents such as halo, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy, aroyl, heteroaroyl, alkoxycarbonyl, alkylcarbonyloxy, nitro, cyano, amino, acyl, sulfonyl, sulfmyl, sulfanyl, sulfoxy, urea, thiourea, sulfamoyl, sulfamide, oxo, carbamoyl, cycloalkyloxy, heterocycloalkyloxy, aryloxy, heteroaryloxy, aralkyloxy, heteroarylalkoxy, or hydroxyl.

[037] As used herein, an "alkynyl" group refers to an aliphatic carbon group that contains 2-8 (e.g., 2-6 or 2-4) carbon atoms and at least one triple bond. Like an alkyl group, an alkynyl group can be straight or branched. An alkynyl group may be optionally substituted with one or more substituents such as halo, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy, aroyl, heteroaroyl, alkoxycarbonyl, alkylcarbonyloxy, nitro, cyano, amino, acyl, sulfonyl, sulfinyl, sulfanyl, sulfoxy, urea, thiourea, sulfamoyl, sulfamide, oxo, carbamoyl, cycloalkyloxy, heterocycloalkyloxy, aryloxy, heteroaryloxy, aralkyloxy, heteroarylalkoxy, or hydroxyl. [038] As used herein, an "amino" group refers to -NR^XR^Y wherein each of R^x and R^γ is independently hydrogen, alkyl, cycloalkyl, (cycloalkyl)alkyl, aryl, aralkyl, heterocycloalkyl, (heterocycloalkyl)alkyl, heteroaryl, or carbonyl each of which are defined herein and are optionally substituted. Examples of amino groups include alkylcarbonylamino, (azacycloalkylcarbonyl)amino, heteroaralkylcarbonylamino, heteroarylcarbonylamino, carbonylamino, (heterocycloalkyl)carbonylamino, (heterocycloalkyl)alkylcarbonylamino, heteroarylcarbonylamino, arylcarbonylamino, aralkylcarbonylamino,

(cycloalkyl)alkylcarbonylamino, cycloalkylcarbonylamino. When the term "amino" is not the terminal group (e.g., alkylcarbonylamino), it is represented by -NR^X-. R^x has the same meaning as defined above. A nonexhaustive list of possible R^x and R^γ includes sulfonylamino, alkylamino, carbonylamino, nitro, carboxy, cyano, oxo, halo, hydroxy, sulfo, mercapto, alkylsulfanyl, alkylsulfinyl, alkylsulfonyl, aminocarbonyl, alkylcarbonyl, cycloalkylcarbonyl, cycloalkylalkylcarbonyl, arylcarbonyl, aralkylcarbonyl, heterocycloalkylcarbonyl, heterocycloalkylalkylcarbonyl, heteroarylcarbonyl or heteroaralkylcarbonyl. [039] As used herein, a "carbonyl" group, when used alone or as part of another structure refers to -(CO)R^X, where R^x is defined above. When the term "carbonyl" is not the terminal group (e.g., arylaminoalkylcarbonyl) it is represented by -C(O)R^X. Without limitation, carbonyl groups can include optionally substituted alkylaminocarbonyl, heterocycloalkenylcarbonyl, (alkylheterocyckOalkenylcarbonyl, heteroarylcarbonyl, arylcarbonyl, heteroarylcarbonyl, alkoxycarbonyl, cycloalkylcarbonyl, alkylheteroarylcarbonyl, arylsulfonylcarbonyl, aminocarbonyl, sulfonylcarbonyl, alkylcarbonyl, alkylsulfonylcarbonyl, alkylcarbonyl or the like. A nonexhaustive list of possible R^x and R^γ includes sulfonylaminocarbonyl, alkylcarbonyl, carbonylamino, carboxy, oxo, hydroxyl, sulfo, mercapto, alkylsulfanyl, alkylsulfinyl, alkylsulfonyl, aminocarbonyl, alkylcarbonyl, cycloalkylcarbonyl, cycloalkylalkylcarbonyl, arylcarbonyl, aralkylcarbonyl, heterocycloalkylcarbonyl, heterocycloalkylalkylcarbonyl, heteroarylcarbonyl, or heteroaralky lcarbonyl .

[040] As used herein, an "aryl" group used alone or as part of a larger moiety as in "aralkyl", "aralkoxy", or "aryloxyalkyl" refers to monocyclic (e.g., phenyl); bicyclic (e.g., indenyl, naphthalenyl, tetrahydronaphthyl, tetrahydroindenyl); tricyclic (e.g., fluorenyl, tetrahydrofluorenyl, anthracenyl, or tetrahydroanthracenyl); or a benzofused group having 3 rings. For example, a benzofused group includes phenyl fused with two or more C₄-₈ carbocyclic moieties. An aryl is optionally substituted with one or more substituents including aliphatic (e.g., alkyl, alkenyl, or alkynyl); cycloalkyl; (cycloalkyl)alkyl; heterocycloalkyl; (heterocycloalkyl)alkyl; aryl; heteroaryl; alkoxy; cycloalkyloxy; heterocycloalkyloxy; aryloxy; heteroaryloxy; aralkyloxy; heteroaralkyloxy; aroyl; heteroaroyl; amino; aminoalkyl; nitro; carboxy; carbonyl (e.g., alkoxycarbonyl, alkylcarbonyl, aminocarbonyl, (alkylamino)alkylaminocarbonyl, arylaminocarbonyl, heteroarylaminocarbonyl, ; or sulfonylcarbonyl); aryalkylcarbonyloxy; sulfonyl (e.g., alkylsulfonyl or aminosulfonyl); sulfmyl (e.g., alkylsulfinyl); sulfanyl (e.g., alkylsulfanyl); cyano; halo; hydroxyl; acyl; mercapto; sulfoxy, urea, thiourea, sulfamoyl, sulfamide, oxo, or carbamoyl. Alternatively, an aryl may be unsubstituted.

[041] Examples of substituted aryls include haloaryl, alkoxycarbonylaryl, alkylaminoalkylaminocarbonylaryl, p, m-dihaloaryl, /7-amino-p-alkoxycarbonylaryl, /w-amino- m-cyanoaryl, aminoaryl, alkylcarbonylaminoaryl, cyanoalkylaryl, alkoxyaryl, aminosulfonylaryl, alkylsulfonylaryl, aminoaryl, />-halo-m-aminoaryl, cyanoaryl, hydroxyalkylaryl, alkoxyalkylaryl, hydroxyaryl, carboxyalkylaryl, dialkylaminoalkylaryl, m- heterocycloaliphatic-o-alkylaryl, heteroarylaminocarbonylaryl, nitroalkylaryl, alkylsulfonylaminoalkylaryl, heterocycloaliphaticcarbonylaryl, alkylsulfonylalkylaryl, cyanoalkylaryl, heterocycloaliphaticcarbonylaryl, alkylcarbonylaminoaryl, hydroxyalkylaryl, alkylcarbonylaryl, aminocarbonylaryl, alkylsulfonylaminoaryl, dialkylaminoaryl, alkylaryl, and trihaloalkylaryl.

[042] As used herein, an "aralkyl" group refers to an alkyl group (e.g., a C_M alkyl group) that is substituted with an aryl group. Both "alkyl" and "aryl" are defined herein. An example of an aralkyl group is benzyl. A "heteroaralkyl" group refers to an alkyl group that is substituted with a heteroaryl. Both "alkyl" and "heteroaryl" are defined herein.

[043] As used herein, a "bicyclic ring system" includes 8-12 (e.g., 9, 10, or 11) membered structures that form two rings, wherein the two rings have at least one atom in common (e.g., 2 atoms in common). Bicyclic ring structures include bicycloaliphatics (e.g., bicycloalkyl or bicycloalkenyl), bicycloheteroaliphatics (e.g., bicycloheteroalkyl or bicycloheteroalkenyl), bicyclic aryls, and bicyclic heteroaryls.

[044] As used herein, a "cycloaliphatic" group encompasses a "cycloalkyl" group and a "cycloalkenyl" group.

[045] As used herein, a "cycloalkyl" group refers to a saturated carbocyclic mono-, bi-, or tri- , or multicyclic (fused or bridged) ring of 3-10 (e.g., 5-10) carbon atoms. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, norbornyl, cubyl, octahydro-indenyl, decahydro-naphthyl, bicyclo[3.2.1]octyl, bicyclo[2.2.2]octyl, bicyclo[3.3.1]nonyl, bicyclo[3.3.2.]decyl, bicyclo[2.2.2]octyl, adamantyl, azacycloalkyl, or (aminocarbonylcycloalkyl)cycloalkyl. A "cycloalkenyl" group, as used herein, refers to a non-aromatic carbocyclic ring of 3-10 (e.g., 4-8) carbon atoms having one or more double bonds. Examples of cycloalkenyl groups include cyclopentenyl, 1,4-cyclohexa- di-enyl, cycloheptenyl, cyclooctenyl, hexahydro-indenyl, octahydro-naphthyl, cyclohexenyl, cyclopentenyl, bicyclo[2.2.2]octenyl, and bicyclo[3.3.1]nonenyl.

[046] A "cycloalkenyl" group, as used herein, refers to a non-aromatic carbocyclic ring of 3- 10 (e.g., 4-8) carbon atoms having one or more double bonds. Examples of cycloalkenyl groups include cyclopentenyl, 1,4-cyclohexa-di-enyl, cycloheptenyl, cyclooctenyl, hexahydro- indenyl, octahydro-naphthyl, cyclohexenyl, cyclopentenyl, bicyclo[2.2.2]octenyl, and bicyclo[3.3.1]nonenyl. Cycloalkenyl ring structures can be optionally substituted at any chemically viable position on the ring or rings.

[047] A cycloalkyl or cycloalkenyl group can be optionally substituted with one or more substituents such as aliphatic (e.g., alkyl, alkenyl, or alkynyl), cycloalkyl, (cycloalkyl)alkyl, heterocycloalkyl, (heterocycloalkyl)alkyl, aryl, heteroaryl, alkoxy, cycloalkyloxy, heterocycloalkyloxy, aryloxy, heteroaryloxy, aralkyloxy, heteroaralkyloxy, aroyl, heteroaroyl, amino, nitro, carboxy, alkoxycarbόnyl, alkylcarbonyloxy, kminocarbonyl, alkylcarbonylamino, cycloalkylcarbonylamino, (cycloalkytyalkylcarbonylamino, arylcarbonylamino, aralkylcarbonylamino, (heterocycloalky^carbonylamino,

(heterocycloalky^alkylcarbonylamino, heteroarylcarbonylaminOj heteroaralkylcarbonylamino, cyano, halo, hydroxyl, acyl, mercapto, sulfonyl (e.g., alkylsulfonyl or arylsulfonyl), sulfinyl (e.g., alkylsulfinyl), sulfanyl (e.g., alkylsulfanyl), sulfoxy, urea, thiourea, sulfamoyl, sulfamide, oxo, carbamoyl, or the like.

[048] Without limitation, examples of substituted cycloaliphatics include alkylcycloalkyl (e.g., propylcyclohexyl), alkylbicyclo[3.1.1]heptyl, alkylcycloalkenyl. [049] As used herein, the term "heterocycloaliphatic" and "heterocyclic" encompasses a heterocycloalkyl group and a heterocycloalkenyl group.

[050] As used herein, a "heterocycloalkyl" group refers to a 3-10 membered mono or bicyclic (fused or bridged) (e.g., 5 to 10 membered mono or bicyclic) saturated ring structure, in which one or more of the ring atoms is a heteroatom (e.g., N, O, S, or combinations thereof). Examples of a heterocycloalkyl group include optionally substituted piperidyl, piperazyl, tetrahydropyranyl, tetrahydrofuryl, 1,4-dioxolanyl, 1,4-dithianyl, 1 ,3-dioxolanyl, oxazolidyl, isoxazolidyl, morpholinyl, thiomorpholyl, octahydro-benzofuryl, octahydro-chromenyl, octahydro-thiochromenyl, octahydro-indolyl, octahydro-pyrindinyl, decahydro-quinolinyl, octahydro-benzo[ό]thiopheneyl, 2-oxa-bicyclo[2.2.2]octyl, l-aza-bicyclo[2.2.2]octyl, 3-aza- bicyclo[3.2.1]octanyl, 2,6-dioxa-tricyclo[3.3.1.0^3>7]nonyl, tropane. A monocyclic heterocycloalkyl group may be fused with a phenyl moiety such as tetrahydroisoquinoline. Heterocycloalkyl ring structures can be optionally substituted at any chemically viable position on the ring or rings.

[051] A "heterocycloalkenyl" group, as used herein, refers to a mono- or bicylic (e.g., 5- to 10-membered mono- or bicyclic) non-aromatic ring structure having one or more double bonds, and wherein one or more of the ring atoms is a heteroatom (e.g., N, O, or S). Examples of heterocycloalkenyls include 2-pyrrolyl, 3-pyrrolyl, 2-imidazolyl, or 2-pyrazolyl. Monocyclic heteroaliphatics are numbered according to standard chemical nomenclature. [052] A heterocycloalkyl or heterocycloalkenyl group can be optionally substituted with one or more substituents such as alkyl (including carboxyalkyl, hydroxyalkyl, and haloalkyl such as trifluoromethyl), alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, heterocycloalkyl (such as a benzimidazolidinyl), (heterocycloalkyl)alkyl, aryl, heteroaryl, alkoxy (two alkoxy groups on the same atom or adjacent atoms may form a ring together with the atom(s) to which they are bound), cycloalkyloxy, heterocycloalkyloxy, aryloxy, heteroaryloxy, aralkyloxy, heteroaralkyloxy, aroyl, heteroairoyl, amino, nitrb, carboxy; alkoxycarbonyl, alkylcarbonyloxy, aminocarbonyl, alkylcarbonylamino, cycloalkylcarbonylamino, (cycloalkyl)alkylcarbonylamino, arylcarbonylamino, aralkylcarbonylamino, (heterocycloalkyl)carbonylamino, (heterocycloalkyl)alkylcarbonylamino, heteroarylcarbonylamino, heteroaralkylcarbonylamino, cyano, halo, hydroxyl, acyl, mercapto, sulfonyl (such as alkylsulfonyl or arylsulfonyl), sulfinyl (such as alkylsulfinyl), sulfanyl (such as alkylsulfanyl), sulfoxy, urea, thiourea, sulfamoyl, sulfamide, oxo, or carbamoyl. [053] A "heteroaryl" group, as used herein, refers to a monocyclic, bicyclic, or tricyclic ring structure having 4 to 15 ring atoms wherein one or more of the ring atoms is a heteroatom (e.g., N, O, S, or combinations thereof) and wherein one or more rings of the bicyclic or tricyclic ring structure is aromatic. A heteroaryl group includes a benzofused ring system having 2 to 3 rings. For example, a benzofused group includes benzo fused with one or two C₄-₈ heterocyclic moieties (e.g., indolizyl, indolyl, isoindolyl, 3H-indolyl, indolinyl, benzo[ό]furyl, benzo[Z>]thiophenyl, quinolinyl, or isoquinolinyl). Some examples of heteroaryl are azetidinyl, pyridyl, lH-indazolyl, furyl, pyrrolyl, thienyl, thiazolyl, oxazolyl, imidazolyl, tetrazolyl, benzofuryl, isoquinolinyl, benzthiazolyl, xanthene, thioxanthene, phenothiazine, dihydroindole, benzo[l,3]dioxole, benzo [b] furyl, benzo[Z>]thiophenyl, indazolyl, benzimidazolyl, benzthiazolyl, puryl, cinnolyl, quinolyl, quinazolyl,cinnolyl, phthalazyl, quinazolyl, quinoxalyl, isoquinolyl, 4H-quinolizyl, benzo-l,2,5-tbiadiazolyl, or 1,8- naphthyridyl.

[054] Without limitation, monocyclic heteroaryls include furyl, thiophenyl, 2H-pyrrolyl, pyrrolyl, oxazolyl, thazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, 1,3,4-thiadiazolyl, 2H-pyranyl, 4-H-pranyl, pyridyl. pyridazyl, pyrimidyl, pyrazolyl, pyrazyl, or 1,3,5-triazyl. Monocyclic heteroaryls are numbered according to standard chemical nomenclature. [055] Without limitation, bicyclic heteroaryls include indolizyl, indolyl, isoindolyl, 3H- indolyl, indolinyl, benzo [b] furyl, benzo [b] thiophenyl, quinolinyl, isoquinolinyl, indolizyl, isoindolyl, indazolyl, benzimidazyl, benzthiazolyl, purinyl, 4H-quinolizyl, quinolyl, isoquinolyl, cinnolyl, phthalazyl, quinazolyl, quinoxalyl, 1,8-naphthyridyl, or pteridyl. Bicyclic heteroaryls are numbered according to standard chemical nomenclature. [056] A heteroaryl is optionally substituted with one or more substituents such as aliphatic including alkyls (e.g., alkoxyalkyl, carboxyalkyl, hydroxyalkyl, oxoalkyl, aralkyl, (alkylsulfonylamino)alkyl, (sulfonylamino)alkyl, cyanoalkyl, aminoalkyl, oxoalkyl, alkoxycarbonylalkyl, (cycloalkyl)alkyl heterocycloalkyl, (heterocycloalkyl)alkyl aralkyl, and haloalkyl such as trifluoromethyl), alkenyl, alkynyl; cycloaliphatic including cycloalkyl (e.g., cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl); heterocycloaliphatic including heterocylcoalkyl (e.g., thiomorpholyl, piperazinyl, 1,3,5-trithianyl, morpholinyl, pyrrolyl, 1,3- dioxolanyl, pyrazolidyl, or piperidinyl); aryl, heteroaryl (e.g., quinolyl, indolyl, 3H-indolyl, isoindolyl, benzo[&]-4H-pyranyl, cinnolyl, quinoxylyl, benzimidazyl, benzo-1,2,5- thiadiazolyl, benzo-l,2,5-oxadiazolyl, or benzthiophenyl); alkoxy; cycloalkyl oxy; heterocycloalkyloxy; aryloxy; heteroaryloxy; aralkyloxy; heteroaralkyloxy; aroyl; heteroaroyl; amino (e.g., carbonylamino, alkylcarbonylamino, alkylsulfonylamino, arylcarbonylamino, cycloalkylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, (heterocycloalkytycarbonylamino, (cycloalkytyalkylcarbonylamino, sulfanylamino, and (heterocycloalkytyalkylcarbonylamino); nitro; carboxy; carbonyl (e.g., alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, arylaminocarbonyl, thiazoleaminocarbonyl, thiomoφholinecarbonyl, aminoalkylaminocarbonyl); alkylcarbonyloxy; cyano; halo; hydroxyl; acyl; mercapto; sulfonyl (e.g., aminosulfonyl, alkylsulfonyl, morpholinesulfonyl. or arylsulfonyl); sulfinyl (e.g., alkylsulfinyl); sulfanyl (e.g., alkylsulfanyl); sulfoxy; urea; • thiourea; sulfamoyl; sulfamide; oxo; or carbamoyl.

[057] Examples of substituted heteroaryls include haloheteroaryl, alkoxycarbonylheteroaryl, alkylaminoalkylaminocarbonylheteroaryl, dihaloheteroaryl, cyanoheteroaryl, aminoheteroaryl, alkylcarbonylaminoheteroaryl, cyanoalkylheteroaryl, alkoxyheteroaryl, aminosulfonylheteroaryl, alkylsulfonylheteroaryl, aminoheteroaryl, aminoheteroaryl, hydroxyalkylheteroaryl, alkoxyalkylheteroaryl, hydroxyheteroaryl, carboxyalkylheteroaryl, dialkylaminoalkylheteroaryl, heterocycloaliphaticheteroaryl, heteroarylaminocarbonylheteroaryl, nitroalkylheteroaryl, alkylsulfonylaminoalkylheteroaryl, heterocycloaliphaticcarbonylheteroaryl, alkylsulfonylalkylheteroaryl, cyanoalkylheteroaryl, heterocycloaliphaticcarbonylheteroaryl, alkylcarbonylaminoheteroaryl, hydroxyalkylheteroaryl, alkylcarbonylheteroaryl, aminocarbonylheteroaryl, alkylsulfonylaminoheteroaryl, dialkylaminoheteroaryl, alkylheteroaryl, and trihaloalkylheteroaryl .

[058] A "heteroaralkyl" group, as used herein, refers to an alkyl group (e.g., a Ci_-4 alkyl group) that is substituted with a heteroaryl group. Both "alkyl" and "heteroaryl" have been defined above.

[059] As used herein, "cyclic moiety" includes cycloalkyl, heterocycloalkyl. cycloalkenyl, heterocycloalkenyl, aryl, or heteroaryl, each of which has been defined previously. [060] As used herein, an "acyl" group refers to a formyl group or alkyl-C(=O)- (also referred to as "alkylcarbonyl") where "alkyl" has been defined previously. Acetyl and pivaloyl are examples of acyl groups.

[061] As used herein, a "carbamoyl" group refers to a group having the structure -O-CO-

NR^XR^Y or -NR^X-CO-O-R^Z wherein R^x and R^γ have been defined above and R^z can be alkyl, aryl, aralkyl, heterocycloalkyl, heteroaryl, or heteroaralkyl.

[062] As used herein, a "carboxy" and a "sulfo" group refer to -COOH or -COOR^X and -

SO₃H or -SO₃R^X, respectively.

[063] As used herein, an "alkoxy" group refers to an alkyl-O- group where "alkyl" has been defined previously. Moreover an alkoxy group includes structures comprising two alkoxy groups on the same atom or adjacent atoms that form a ring together with the atom(s) to which they are bound.

[064] As used herein, a "sulfoxy" group refers to -O-SO-R^X or -SO-O-R^X, where R^x has been defined above.

[065] As used herein, a "mercapto" group refers to -SH.

[066] As used herein, a "sulfonyl" group refers to -S(O)₂-R^X, wherein R^x has been defined above. Examples of sulfonyls include alkylsulfonyl or arylsulfony.

[067] As used herein a "sulfinyl" group refers to -S(O)-R^X, wherein R^x has been defined above. Examples of sulfinyls include alkylsulfϊnyl.

[068] As used herein a "sulfanyl" group refers to -S-R^x, wherein R^x has been defined above.

Examples of sulfanyl s include alkylsulfanyl.

[069] As used herein, a "halogen" or "halo" group refers to fluorine, chlorine, bromine or iodine.

[070] As used herein, a "haloaliphatic" group refers to an aliphatic group substituted with 1 -3 halogen. For instance, the term haloalkyl includes the group -CF₃.

[071] As used herein, a "sulfamoyl" group refers to the structure -S(O)₂-NR^XR^Y or -NR^X -

S(O)₂-R² wherein R^x, R^γ, and R^z have been defined above.

[072] As used herein, a "sulfamide" group refers to the structure -NR^X -S(O)₂-NR^YR^Z wherein R^x, R^γ, and R^z have been defined above.

[073] As used herein, a "carbonylamino" group used alone or in connection with another group refers to an amido group such as R -C(O)-NR -. For instance an alkylcarbonylamino includes alkyl-C(O)-NR^x-, wherein R^x has been defined above.

[074] As used herein, a "aminocarbonyl" group used alone or in connection with another group refers to an amido group such as N(R )₂-C(O)-. [075] As used herein, an "alkox^'ycarbonyl" used alone or in connection with another group refers to a carbonyl group such as alkyl-O-C(O)-.

[076] As used herein, an "alkoxyalkyl" refers to an alkyl group such as alkyl-O-alkyl-, wherein alkyl has been defined above.

[077] As used herein, an "aminocarbonyl" refers to an amido group such as -NR^X-C(O)-, wherein R has been defined above.

[078] As used herein, an "aminosulfonyl" refers to the structure -N(R^X)₂-S(O)₂-, wherein R^x has been defined above.

[079] As used herein, an "oxo" refers to =0.

[080] As used herein, an "aminoalkyl" refers to the structure N(R^x)₂-alkyl-.

[081] used herein, a "cyanoalkyl" refers to the structure (CN)-alkyl-.

[082] As used herein, an "alkylsulfonyl" group refers to the structure alkyl-S(O)₂.

[083] As used herein, a "sulfonylamino" group refers to the structure R^X-S(O)₂-N(R^X)₂-, wherein R^x has been defined above.

[084] As used herein, a "urea" group refers to the structure -NR^X-CO-NR^YR^Z and a

"thiourea" group refers to the structure -NR^X-CS-NR^YR^Z. R^x, R^γ, and R^z have been defined above.

[085] The phrase "optionally substituted" is used interchangeably with the phrase "substituted or unsubstituted." As described herein, compounds of the invention can optionally be substituted with one or more substituents, such as are illustrated generally above, or as exemplified by particular classes, subclasses, and species of the invention. As described herein, the variables Ri, R2, R₃, and R4, and other variables contained therein formulae I encompass specific groups, such as alkyl and aryl. Unless otherwise noted, each of the specific groups for the variables Ri, R₂, R₃, and R₄, and other variables contained therein can be optionally substituted with one or more substituents described herein. Each substituent of a specific group is further optionally substituted with one to three of halo, cyano, oxoalkoxy, hydroxyl, amino, nitro, aryl, haloalkyl, and alkyl. For instance, an alkyl group can be substituted with alkylsulfanyl and the alkylsulfanylcan be optionally substituted with one to three of halo, cyano, oxoalkoxy, hydroxyl, amino, nitro, aryl, haloalkyl, and alkyl. As an additional example, the cycloalkyl portion of a (cycloalkyl)carbonylamino can be optionally substituted with one to three of halo, cyano, alkoxy, hydroxyl, nitro, haloalkyl, and alkyl.

[086] In general, the term "substituted," whether preceded by the term "optionally" or not, refers to the replacement of hydrogen radicals in a given structure with the radical of a specified substituent. Specific substituents are described above in the definitions and below in the description of compounds and examples thereof. Unless otherwise indicated, an optionally substituted group may have a substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. A ring substituent, such as a heterocycloalkyl, may be bound to another ring, such as a cycloalkyl, to form a spiro-bicyclic ring system, e.g., both rings share one common atom. Combinations of substituents envisioned by this invention are those combinations that result in the formation of stable or chemically feasible compounds.

[087] The phrase "stable or chemically feasible," as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and preferably their recovery, purification, and use for one or more of the purposes disclosed herein. In some embodiments, a stable compound or chemically feasible compound is one that is not substantially altered when kept at a temperature of 40⁰C or less, in the absence of moisture or other chemically reactive conditions, for at least a week. [088] Unless otherwise stated, structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure; for example, the R and S configurations for each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the invention. Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention. Additionally, unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a ¹³C- or ^I4C-enriched carbon are within the scope of this invention. Such compounds are useful, for example, as analytical tools or probes in biological assays.

[089] As used herein, a "detectable quantity" means that the amount of the detectable compound that is administered is sufficient to enable detection of binding of the compound to a biofilm.

[090] As used herein, an "imaging effective quantity" means that the amount of the detectable compound that is administered is sufficient to enable imaging of binding of the compound to a biofilm. [091] The term "in vivo imaging" refers to any method that permits the detection of a labeled compound in a live animal.

[092] As used herein, the term "subject" refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, rodents, and the like, which is to be the recipient ' of a particular treatment. Typically, the terms "subject" and "patient" are used interchangeably herein in reference to a human subject.

[093] As used herein, the term "animal" refers to all animals including, but are not limited to, vertebrates such as rodents, non-human primates, ovines, bovines, ruminants, lagomorphs, porcines, caprines, equines, canines, felines, aves, etc.

II Biofilm Assay

[094] The assay uses fluorescent compounds to detect biofilms or biofilm formation by microorganisms.

A Fluorescent compounds

[095] The compounds used in the biofilm assay fluoresce when the compounds bind to or interact with the PIA of the biofilm produced and/or secreted by microorganisms. Several classes of fluorescent compounds were found to specifically label the PIA of Staphylococcus species and other species of microorganisms that synthesize PIA or similar polysaccharide molecules. The selective compounds also produce selective fluorescence emission by providing an increased level of fluorescence emission upon contacting and interacting with PIA from biofilms or microorganisms relative to the level of fluorescence emission produced by the compound or compositions alone. Fluorescent compounds that selectively detect PIA in biofilms or from microorganisms have a number of advantages: specificity for detecting biofilms formed by specific microorganisms capable of producing PIA or similar polysaccharides, high sensitivity resulting from the selective fluorescent emission, facilitation of high throughput screening and real time monitoring.

1. Description of the Compounds

[096] The fluorescent compounds useful in conducting the biofilm assay are from three major chemical classes: thioflavins and thioflavin derivatives, styrylbenzenes and styrylbenzene derivatives and napthalene derivatives. i. Thioflavins and Thioflavin Derivatives [097] In one aspect, the selectively fluorescent compounds have the structure of formula I.

I or a salt thereof, wherein X is S₅ O or -CH;

Y is C or N; if Y is C, the bond represented by the dashed line is a bond to form a double bond; if

Y is N, the bond represented by the dashed line is absent. Ri is H, or a lower alkyl group;

R₂ is H₃ a lower alkyl group, -OH, -CO₂H, -CH₂CO₂H, -NH₂, -NHCH₃, 1, F, Cl, Br,

R₃ is H, I, F, Cl or Br;

R_t is H, -CH₃, or absent; if R₄ is H or -CH3, a quaternary nitrogen is formed; R₅ is -NH₂, -NHCH₃, -N(CH₃)CH₃, -OH, -OCH₃, or

|-N N-CH₃ ^ — / ; and

R₆ is H, I, F, Cl or Br.

[098] In an embodiment, the method of detecting a biofilm includes contacting microorganisms or a biofilm with a benzathiazole of formula II

Il or a salt thereof, wherein

R₇ is H, a lower alkyl group, -OH, -CO₂H, -CH₂CO₂H, I, F, Cl or Br;

R₈ is H, -CH₃, or absent; and

R₉ is -NH₂, -NHCH₃, -N(CH₃)CH₃, -OH, -OCH₃, or

|-N N-CH₃ [099] In another embodiment, the method of detecting a biofϊlm includes contacting microorganisms or a biofilm with a benzathiazole of formula III.

III or a salt thereof, wherein

R₁₀ is H₃ a lower alkyl group, -OH, -CO₂H, -CH₂CO₂H, I, F, Cl or Br;

R_n is H, F₅ Cl, Br, I, -SO₃ ^", -SH, -NO₂, -NH₂, or a lower alkyl group;

R₁₂ is H, -CH₃, or absent; and

R₁₃ is -NH₂, -NHCH₃, -N(CH₃)CH₃, -OH, or -OCH₃.

[0100] In yet another embodiment, the method of detecting a biofilm includes contacting microorganisms or a biofϊlm with a benzathiazole of formula IV

or a salt thereof, wherein

Ri₄ is H, a lower alkyl group, -OH, -CO₂H, -CH₂CO₂H, -NH₂, -NHCH₃, 1, F, Cl or Br and

R₁₅ is -NH₂, -NHCH₃, -N(CH₃)CH₃, -OH, or -OCH₃.

[0101] In a further embodiment, the method of detecting a biofilm includes contacting microorganisms or a biofilm with a benzoxazole of formula V

V or a sale thereof, wherein

R₁₆ is I, F, Cl, or Br and

Ri₇ and Rjg are independently H, -CH₃, Or-CHaCH₃.

[0102] In yet another embodiment, the method of detecting a biofilm includes contacting microorganisms or a biofilm with an imadazopyridine of formula VI

VI or a pharmaceutically acceptable salt thereof, wherein

Ri₉ is H, a lower alkyl group or I, Br, F, or Cl and

R_2O and R₂₁ are independently H, -CH₃, or -CH₂CH₃.

[0103] In yet another embodiment, the method of detecting a biofilm includes contacting microorganisms or a biofilm with either Thioflavin-T or Thioflavin-S. Thiofiavin T has the formula:

Thiofiavin S is a mixture of compounds, with the major component having the formula

[0104] Other thiofiavin derivatives useful in detecting biofilms are disclosed in International Publication Nos. WO 02/16333A2, WO 02/075318A2 and WO 04/083195A1, each of which is herein incorporated by reference. ii. Styrylbenzenes

[0105] In a second aspect, the method of detecting a biofilm includes contacting microorganisms or a biofilm with a styrylbenzene or a styrylbenzene derivative of formula VII

VII or a salt thereof, wherein

R₂₂- is -OH, or -0(Ci-C₄ alkyl); R₂₃ is -CO₂Me, -CO₂H or -OH; and R₂₄ is Br, I, F, Cl, H, or -O(d-C₄ alkyl). [0106] In an embodiment, the method of detecting a biofilm includes contacting microorganisms or a biofilm with a styrylbenzene or a styrylbenzene derivative of formula VII, wherein R₂₂ is -OH or -OCH₃, R₂₃ is -CO₂H or -CO₂Me, and R₂₄ is Br.

[0107] In a second embodiment, the method of detecting a biofilm includes contacting microorganisms or a biofilm with the styrylbenzene or a styrylbenzene derivative of formula

VII wherein R₂₂ is -OH or -OCH₃, R₂₃ is -CO₂H, and R₂₄ is Br, I, H or -OCH₃.

[0108] In yet another embodiment, the method of detecting a biofilm includes contacting microorganisms or a biofilm with a styrylbenzene of formula VIII

VIII or a salt thereof, wherein R2₅ is either I, Br₅ or F. iii. Napthalene Derivatives

[0109] In a third aspect, the method of detecting a biofilm includes contacting microorganisms or a biofilm with a napthalene derivative of formula IX

R26 n JL ^"3 ^K27

IX or a salt thereof, wherein

R₂₆ is-OCH₃, -N(CH₃)CH₂CH₂F, -N(CH₃)CH₂CH₂I, -N(CH₃)CH₂CH₂Br, and

[0110] In an embodiment, the napthalene derivative has the formula X

X or a salt thereof, wherein

R₂₈ is-OCH₃, -N(CH₃)CH₂CH₂F, -N(CH₃)CH₂CH₂I, -N(CH₃)CH₂CH₂Br, and

[0111] Other compounds that exhibit selective fluorescence are disclosed in International Publication No. WO 02/07781 A2₇ which is incorporated herein by reference. [0112] The above described compounds have a number of advantages over other compounds, particularly other compounds which are used to detect biofilms or microorganisms using absorption techniques.

[0113] First, compounds that selectively fluoresce upon interacting with PIA do not provide selective fluorescence when interacting with biofilms produced by Pseudomonas species and other microorganisms which do not produce PIA or similar polysaccharide molecules. Consequently, the method of this invention has the advantage of specifically detecting Staphylococcus species and other microorganisms that synthesize PIA or a similar structures. This allows one to distinguish between biofilms made by Pseudomonas species and those made by Staphylococcus species or other microorganisms that synthesize PIA. [0114] Second, the fluorescent compounds used in the method provide a higher sensitivity over absorption methods that use absorbance compounds such as crystal violet and saffranin. The selectively fluorescent compounds exhibit increased levels of fluorescence emission when interacting with PIA and similar molecules relative to the fluorescence emission of the compounds alone. In some embodiments, the intensity of fluorescence emission by the compound alone is about 10 fold or more less than the intensity of fluorescence emission from the compound when it interacts with the biofilm or microorganisms producing PIA or similar polysaccharides. The difference in the intensity between a background fluorescence signal, when the compound is not interacting with PIA, versus the intense fluorescence signal resulting when the compound interacts with PIA provides a highly selective and sensitive technique for detecting the presence of and measuring the amount of biofilm or the microorganisms that create biofilms.

[0115] The sensitivity of the method is related to the ratio- of the fluorescence emission signal to the noise in the fluorescence detection system ( e.g., signal-to-noise). The selectively fluorescent compounds provide intense fluorescence signals which are above the noise level of the detection systems. For instance, Thioflavin-T produces an intense fluorescence signal in which the noise can be measured as less than 5% of the signal intensity. Whereas, absorption techniques could have noise levels as high as 20-30% of the signal. The assays utilizing the selectively fluorescent compounds with increased signal to noise permit detection of a 2-5% difference between samples without the need to average multiple, such as hundreds or thousands, spectra. Consequently, use of the fluorescent compounds of this invention results in a 10 fold greater sensitivity for measuring biofilms. [0116] A third advantage is the ability to use much less dye in the method. The use of Thioflavin T requires 1000 fold less dye than absorption assays utilizing dyes that absorb in the visible spectrum.

[0117] A fourth advantage is ease of handling for high throughput screening. Absorbance assays that use dyes, such as crystal violet and saffranin, require multiple wash steps to remove unbound dye prior to recording before the absorption spectra. The fluorescent compounds emit increased levels of fluorescence when contacting or interacting with a biofilm, whereas the unbound compound emits less or no fluorescence. Accordingly, there is little or no need to wash out the unbound compound, resulting in fewer steps for high throughput screening. The procedure can be performed in as little as three steps: 1) adding the fluorescent compound, 2) decanting the liquid and 3) reading the fluorescence.

[0118] As a fifth advantage, the method provides real-time monitoring of biofilm formation as a result of the strong fluorescent signal, the low amount of compound needed, and selective fluorescence.

2. General Synthetic Methodology

[0119] The compounds useful for measuring biofilms can be obtained from commercial sources or can be prepared in general by methods known to those skilled in the art for analogous compounds. The starting materials for producing the compounds are commercially available from chemical reagent supply companies, such as Sigma-Aldrich Company, Fisher Scientific, Calbiochem, and the like. Compounds that are not commercially available can be prepared by those of ordinary skill in the art following procedures set forth in references such as "Fieser and Fieser's Reagents for Organic Synthesis", Volumes 1-23, John Wiley and Sons, 2006; "Rodd's Chemistry of Carbon Compounds", Volumes 1-5 and Supplements, Elsevier Science Publishers, 1997; and "Organic Reactions", Volumes 1-68, John Wiley and Sons, 2007.

[0120] Thioflavins and thioflavin derivatives are available either commercially, or can be prepared by those of ordinary skill in the art. Thioflavin-T is available from Sigma-Aldrich Co., and Thioflavin-S can be obtained from Fisher Chemicals or from Chemos GmBH. Methods for preparing compounds of formulas I- VI are set forth in International Publication Nos. WO 02/16333A2, and WO 04/083195A1 and in Klunk, et ah, Life Sci.(1991) 69:1471- 84, which are herein incorporated by reference. Methods for preparing compounds of formulas II and IV are set forth in Zhuang et al., J. Med. Chem. (2001), 44:1905-14, which is herein incorporated by reference. One of the compounds of formula IV, wherein R₁ is H and R₂ is — NHCH₃ is available from Sigma- Aldrich. Another compounds of formula IV, wherein R₁ is — OH and R₂ is -NHCH₃ is disclosed in Klunk, et al., Ann. Neurol. (2004) 55:306-319, which is herein incorporated by reference. Methods for preparing compounds of formula V are detailed in Shuang, et al, Nucl. Med. Biol. (2001) 28:887-894, and methods for preparing compounds of formula VI are set forth in Kung, et al. (2002) Brain Res. 956: 202-10. both of which are herein incorporated by reference.

[0121] Methods for preparing styrylbenzenes of formula VII are set forth in Zhuang, et al. (2001) J. Med. Chem., 44: 1905-14 and Lee et al. (2001) J. Med. Chem., 44: 2270-75, which are herein incorporated by reference. Methods for preparing the compounds of formula VIII are set forth in Kung, et al. (2001) J. Am. Chem. Soc. 123: 127 '40-12741, which is herein incorporated by reference.

[0122] Methods to prepare the napthalene derivatives of formulas IX and X are set forth in Agdeppa, et al. (2001) J. Neurosci., 21 RC 189: 1-5, which is herein incorporated by reference. The compound of formula X wherein Ri is -OCH₃ and R₂ is -C(CH₃)COaH is available from Sigma-AIdrich.

B. Microorganisms

[0123] The method can be used to detect the presence of certain microorganisms that form biofilms. Because a biofilm is measured using molecules that fluoresce when bound to PIA or a similar polysaccharide structure, the assay can be used to detect the presence of any microorganism that produces PIA or a similar polysaccharide.

[0124] In one embodiment, the microorganism is Staphylococcus aureus or Staphylococcus epidermidis. The microorganisms can also be other Staphylococcus species that produce PIA. These could include Staphylococcus epidermis, Staphylococcus aureus, Staphylococcus haemolyticus, Staphylococcus saprophyticus, Staphylococcus hominis, Staphylococcus warneri, Staphylococcus cohnii, Staphylococcus capitis, Staphylococcus camosis, Staphylococcus lugdunensis, other Staphylococcus species or a combination thereof. [0125] In another embodiment, the microorganism is an Escherichia coli strain that produces PIA or a similar molecule. In a third embodiment, the microorganism is Yersinia pestis, which produces PIA.

[0126] The microorganisms can be a pure culture containing only one type of microorganism. Alternatively, the culture may include a mixture of strains and/or species of microorganisms. The species may be known and characterized, unknown, or a mixture of known and unknown species. In some embodiments, the culture system comprises raw environmental samples from pristine environments (e.g., from soil, aquatic, rhizosphere, rhizoplane), from human-impacted environments such as toxic sites, industrial sites, agriculture, waste water treatment plants, and/or from animals or humans, such as oral samples, or samples from other parts of the body, or from household or industrial environments.

C. Vessels

[0127] The method for detecting microorganisms and/or biofilms can be performed in a number of vessels including, but not limited to, test tubes, culture dishes, petri plates, beakers, flasks, cups, multiwell plates or any container that can hold liquid or semi-solid media. [0128] In one embodiment, the vessel is a multiwell plate. In a further embodiment, the multiwell plate contains 96, 384 or more wells. Because the dyes fluoresce only when contacting the biofilm, wash steps are not required, unlike assays using absorbance dyes. Thus, this assay is well suited for high throughput screening using multiwell plates with 384 or more wells.

[0129] The vessel can also be an apparatus designed to grow and measure biofilms that form on the surfaces, including the surface of a liquid. These vessels contain biofilm adherent sites, which can be projections extending from a surface of a vessel. The biofilm adherent sites can also be studs that can be removed to examine biofilm growth. The vessel can hold medium for biofilm growth so that the projections are in contact with the medium. [0130] In one embodiment, the apparatus can include an upper plate that includes projections and a lower plate containing a plurality of wells or troughs. During operation, the wells in the lower plate contain growth medium and the upper plate is configured to position projections into each of the wells.

[0131] In another embodiment, the apparatus includes a tube through which growth media can flow. The tube can have a plurality of ports within the tube wall, with each port having a removable stud, which has a surface upon which biofilms can grow. The stud can be removed and analyzed for growth of microorganisms.

[0132] Examples of such apparatuses include without limitation the Calgary peg plate described in US Patent No. 6,596,505,. the Robins device described in the Canadian Journal of Microbiology (1981), 27:910-927, and the apparatus described in U.S. Pat. No. 5,349, 874. [0133] The vessel can also include a medical device or portion thereof, including, without limitation, a catheter, a stent or a valve.

[0134] The vessels can be composed of a wide variety of materials including, without limitation, glass, polypropylene, polystyrene, polyvinylchloride, or other plastics, silicone, Teflon, or other materials used in medical devices. The vessel can also be made of a biomaterial, such as enamel or a dental materials or an acellular artery or valve from a human or other animal.

[0135] The vessels can be coated with a biotic material. The biotic material may be lysine or polylysine, which is present in TC plates and flasks. In addition to lysine or polylysine, the vessels can also be coated with another biotic coating, including, without limitation, serum proteins, albumin, mucin, polyurethane or hyaluronic acid.

D. Other Components

[0136] Other components of the method include a growth medium. [0137] The growth medium can be any liquid, semi-solid or solid media on which microorganisms can grow. The media include without limitation, rich media formulated for the microorganism, such as Tryptic soy broth with or without added glucose or other carbon source; media taken from an environment in which a biofϊlm could grow, including body fluids and tissues, or materials found in a specific environment, such as a pond, materials and fluids in household pipes, materials and fluids in industrial equipment, or materials from air samples. [0138] The incubation time can be as short a nanosecond to as long as a number of months. Typically, for Staphylococcus species, the incubation time is about 16-20 hours.

E. Methods for Detecting Fluorescence

[0139] Fluorescence can be detected using any instrument that can detect fluorescence. The instrument can be, without limitation, a spectrafluorometer that can accept a number of vessels including test tubes and multiwell plates. Surface fluorescence can be detected and quantitated using fiber optic technology that delivers excitation irradiation and detects fluorescence emission. Other techniques for detection include without limitation fluorescent, laser-confocal and cross-polarization microcopy.

HI Screening Method for Compounds that Modulate Bioiϊlms [0140] In another aspect, the invention provides a method for identifying compounds that modulate biofilms or biofϊlm formation. The method includes contacting microorganisms with a compound and measuring fluorescence using a thioflavin, a thioflavin derivative, a styrylbenzene, a styrylbenzene derivative or a napthalene derivative. A measurable difference in binding of the fluorescent compounds in the presence and absence of the potential modulator indicates a compound that modulates the biofϊlm or biofϊlm formation. A decrease in fluorescence would indicate a compound that decreases a biofilm or biofϊlm formation. Alternatively, an increase in fluorescence would indicate a compound that increases a biofilm or biofilm formation.

[0141] Like the method for measuring biofilms without the added modulator, the screening method can be used to screen for compounds that modulate biofilms or biofilm formation by microorganisms that produce PIA or polysaccharide compound. The microorganisms include without limitation, S. aureus, S. epidermidis, some strains of E. coli, and Y. pestis. The vessels may be any vessels described previously and include test tubes, multiwell plates, culture dishes and flasks, beakers and medical devices or portions thereof. The vessels may be composed of a variety of materials as described above. The vessels may also be coated with a biotic coating or be provided with a surface material as described above. Any of the fluorescent compounds described above may be used to measure the biofilm.

[0142] A variety of reagents may be used to modify conditions for biofilm formation. These include, without limitation, varied salt and carbon source concentrations, and varying the components and concentrations of the growth medium. Environmental conditions may also be varied, including pH, incubation time, temperature, osmolality and other variables. [0143] The screening method can also be used to identify compounds that can kill microorganisms in a biofilm form. After a biofilm is formed it can be treated with a compound for a period of time. After treatment, medium is removed and replaced with fresh medium. The cells in the biofilm can then be disrupted and tested for viability or ability to form another biofilm. The fluorescent compounds can be used as an initial screen to determine loss of biofilm due to cell death.

IV Kits for Biofilm Assays

[0144] The materials for use in the method of the invention are ideally suited for the preparation of a kit. Such a kit may comprise a carrier means being compartmentalized to receive one or more container means such as vials, tubes, and the like, each of the container means comprising one of the separate elements to be used in the method. For example, one of the container means may comprise one or more fluorescent compounds, either a thioflavin, a thioflavin derivative, a styrylbenzene. a styrylbenzene derivative, a napthalene derivative or a combination thereof. A second container may further comprise growth medium. A third container may comprise microorganisms. Constituents may be present in liquid or lyophilized form, as desired.

V. In Vivo Imaging [0145] The method can also be used to detect the presence and location of biofilms in an organ or body area of an animal or patient. The method comprises administering a detectable quantity of a fluorescent compound, such as a pharmaceutical composition containing one of the fluorescent compounds, to an animal or patient.

[0146] The method of in vivo imaging utilizes the fluorescent compounds in conjunction with non-invasive imaging techniques such as magnetic resonance spectroscopy (MRS) or imaging (MRI), or gamma imaging such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT) to detect and/or quantify biofilm formation in vivo. [0147] For gamma imaging, the radiation emitted from the organ or area being examined is measured and expressed either as total binding or as a ratio in which total binding in one tissue is normalized to (for example, divided by) the total binding in another tissue of the same subject during the same in vivo imaging procedure. Total binding in vivo is defined as the entire signal detected in a tissue by an in vivo imaging technique without the need for correction by a second injection of an identical quantity of labeled compound along with a large excess of unlabeled, but otherwise chemically identical compound. [0148] For purposes of in vivo imaging, the type of detection instrument available is a factor in selecting a specific label. For instance, radioactive isotopes and ¹⁹F are particularly suitable for in vivo imaging. The type of instrument used will guide the selection of the radionuclide or stable isotope. For example, the radionuclide chosen must have a type of decay detectable by a given type of instrument. In addition, the half-life of the radionuclide should be long enough so that it is still detectable at the time of maximum uptake by the target, but short enough so that the host does not sustain deleterious radiation. Radiolabeled compounds can be detected using gamma imaging wherein emitted gamma irradiation of the appropriate wavelength is detected. Methods of gamma imaging include, but are not limited to, SPECT and PET. In one embodiment, for SPECT detection, the chosen radiolabel will lack a particulate emission, but will produce a large number of photons in a 140-200 keV range. For PET detection, the radiolabel will be a positron-emitting radionuclide such as ¹⁹F which will annihilate to form two 511 keV gamma rays which will be detected by the PET camera. [0149] The thioflavins, thiofiavin derivatives, styrylbenzenes, styrylbenzene derivatives and the napthalene derivatives can be labeled with ¹⁹F or ¹³C for MRS/MRI by general organic chemistry techniques known to the art. See, e.g., March, J. "ADVANCED ORGANIC CHEMISTRY: REACTIONS, MECHANISMS, AND STRUCTURE (6th Edition, 2007), the contents of which are hereby incorporated by reference. The compounds can also be radiolabeled with ¹⁸F, ¹¹C, ⁷⁵Br, or ⁷⁶Br for PET by techniques well known in the art and are described by Fowler, J. and Wolf, A. in POSITRON EMISSION TOMOGRAPHY AND AUTORADIOGRAPHY (Phelps, M., Mazziota, J., and Schelbert, H. eds.) 391-450 (Raven Press, N. Y. 1986) the contents of which are hereby incorporated by reference. The compounds can also be radiolabeled with ¹²³I for SPECT by any of several techniques known to the art. See, e.g., Kulkarni, Int. J. Rad. Appl. & Inst. (Part B) 18: 647 (1991), the contents of which are hereby incorporated by reference. Additionally, the thioflavins, thioflavin derivatives, styrylbenzenes, styrylbenzene derivatives and the napthalene derivatives can be labeled with any suitable radioactive iodine isotope, such as, but not limited to ¹³¹1, ¹²⁵I₅ or ¹²³I, by iodination of a diazotized amino derivative directly via a diazonium iodide (see Greenbaum, F. Am. J. Pharm. 108: 17 (1936)) or by conversion of the unstable diazotized amine to the stable triazene, or by conversion of a non-radioactive halogenated precursor to a stable tri-alkyl tin derivative which then can be converted to the iodo compound by several methods well known to the art. See, Satyamurthy and Barrio J. Org. Chem. 48: 4394 (1983), Goodman et al., J. Org. Chem. 49: 2322 (1984), and Mathis et al., J. Labell. Comp. and Radiopharm. 1994: 905; Chumpradit et al., J. Med. Chem. 34: 877 (1991); Zhuang et al., J. Med. Chem. 37: 1406 (1994); Chumpradit et al., J. Med. Chem. 37: 4245 (1994). For example, a stable triazene or tri-alkyl tin derivative of a thioflavin, a thioflavin derivative, a styrylbenzene, a styrylbenzene derivative or a napthalene derivative is reacted with a halogenating agent containing ¹³¹I, ¹²⁵I, '¹²³I, ⁷⁶Br, ⁷⁵Br, ¹⁸F or ¹⁹F. Thus, the stable triazene and tri-alkyl tin derivatives of the compounds can be precursors useful for the synthesis of many of the radiolabeled compounds used in the present invention.

[0150J The thioflavins, thioflavin derivatives, styrylbenzenes, styrylbenzene derivatives and the napthalene derivatives also can also be radiolabeled with known metal radiolabels, such as Technetium-99 m (" ^mTc). Modification of the substituents to introduce ligands that bind such metal ions can be effected without undue experimentation by one of ordinary skill in the radiolabeling art. The metal radiolabeled compounds can then be used to detect biofilms. [0151] The methods of the present invention may use isotopes detectable by nuclear magnetic resonance spectroscopy for purposes of in vivo imaging and spectroscopy. Elements particularly useful in magnetic resonance spectroscopy include ¹⁹F and ¹³C. [0152] Suitable radioisotopes for the in vivo methods include beta-emitters, gamma-emitters, positron-emitters and x-ray emitters. These radioisotopes include ¹³¹1, ¹²³1, ¹⁸F, ¹¹C, ⁷⁵Br and ⁷⁶Br. Suitable stable isotopes for use in Magnetic Resonance Imaging (MRI) or Spectroscopy (MRS)₅ include ¹⁹F and ¹³C. In some embodiments, radiolabels are ¹⁸F for use in PET in vivo imaging, ¹²³I for use in SPECT imaging and ¹⁹F for MRS/MRI. [0153] For in vivo imaging, the compounds are formulated into a pharmaceutical composition. A typical composition comprises a pharmaceutically acceptable carrier. For instance, the composition may contain about 10 mg of human serum albumin and from about 0.5 to 500 micrograms of a labeled thioflavin, a thioflavin derivative, a styrylbenzene, a styrylbenzene derivative or a napthalene derivative per milliliter of phosphate buffer containing NaCl. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like, as described, for instance, in REMINGTON'S PHARMACEUTICAL SCIENCES, 18^th Edition, Easton: Mack Publishing Co. (1995) and UNITED STATES PHARMACOPEIA, 30^th Edition, NATIONAL FORMULARY 25^th Edition, U.S. Pharmacopeia (2007), the contents of which are herein incorporated by reference. [0154] Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobials, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components of the pharmaceutical composition are adjusted according to routine skills in the art. See, Goodman and Gilman's THE PHARMACOLOGICAL BASIS FOR THERAPEUTICS (1 lth Ed., 2005), McGraw-Hill Co., Professional, the contents of which are incorporated herein by reference. [0155] In one embodiment, pharmaceutical compositions are those that, in addition to specifically binding biofilms in vivo, are also non-toxic at appropriate dosage levels and have a satisfactory duration of effect.

[0156] The method could also be used to diagnose an infection in patients or monitor a patient for infection, particularly those at risk for infections, such as immunocompromised patients. This method can also be used to monitor the effectiveness of therapies targeted at treating and/or preventing biofilm formation.

[0157] Generally, the dosage of the detectably labeled thioflavins, thioflavin derivatives, styrylbenzenes, styrylbenzene derivatives and the napthalene derivatives will vary depending on considerations such as age, weight and the particular region of the body to be scanned, and the imaging agent used. Other variables include the condition, sex, and extent of disease in the patient, contraindications, if any, concomitant therapies and other variables, to be adjusted by a physician skilled in the art. Dosage can vary from 0.001 mg/kg to 1000 mg/kg or from 0.1 mg/kg to 100 mg/kg. [0158] Administration to the subject can be topical, oral, pulmonary, intranasal, intraperitoneal or parenteral. Parenteral administration includes administration by the following routes: intravenous, intramuscular, interstitially, intra-arterially, subcutaneous, intraocular, intrasynovial, transepithelial, including transdermal, pulmonary via inhalation, ophthalmic sublingual and buccal, topically, including ophthalmic, dermal, ocular, rectal, and nasal inhalation via insufflation.

[0159] After a sufficient time has elapsed for the compound to bind with the biofϊlm, for example 30 minutes to 48 hours, the area of the subject under investigation is examined by routine imaging techniques such as MRS/MRI, SPECT, planar scintillation imaging, PET, as well as emerging imaging techniques. The exact protocol will necessarily vary depending upon factors specific to the patient, as noted above, and depending upon the body site under examination, method of administration and type of label used; the determination of specific procedures would be routine to the skilled artisan.

VI In Vitro Imaging

[0160] The method can be used to detect and/or measure biofilms in biopsy or post-mortem tissue. The method involves incubating tissue with a solution of a thioflavin, a thioflavin derivative, a styrylbenzene, a styrylbenzene derivative or a napthalene derivative described above. In one embodiment, the solution is 25-100% ethanol (with the remainder being water) saturated with a thioflavin, a thioflavin derivative, a styrylbenzene, a styrylbenzene derivative or a napthalene derivative. Upon incubation the compound stains or labels the biofϊlm in the tissue and the stained or labeled deposit can be detected or visualized by any standard method, including without limitation, fluorescent, laser-confocal and cross-polarization microscopy. [0161] The method of quantifying the amount of biofϊlm in biopsy or post-mortem tissue involves incubating a labeled thioflavin, thioflavin derivative, styrylbenzene, styrylbenzene derivative, napthalene derivative or a water-soluble, non-toxic salt thereof, with homogenate of biopsy or post-mortem tissue. The tissue is obtained and homogenized by methods well known in the art. The amount of biofϊlm can be measured by the fluorescence of the compound, or by other methods if the compound is labeled, for example, with a radiolabel. If the compound is radiolabeled, labeled with an enzyme, a chemiluminescent or immunofluorescent label, the biofϊlm can be detected by radioactivity, enzyme activity, luminescence or immunofluorescence. The radiolabel can include ¹²⁵1, ¹⁴C and ³H. Tissue containing biofϊlm will bind to the labeled compounds. The bound tissue is then separated from the unbound tissue by any mechanism known to the skilled artisan, such as filtering. The bound tissue can then be quantified by any means known to the skilled artisan. The units of tissue-bound labeled compound are then converted to units of micrograms of biofilm (or PIA) per 100 mg of tissue by comparison to a standard curve generated by incubating known amounts of biofilm (or PIA) with the thioflavin derivative.

[0162] Other embodiments are described in the following non-limiting examples.

EXAMPLES

EXAMPLE 1 : Bacterial strains and growth conditions

[0163] Permanent frozen stocks of all bacteria were maintained at -8O⁰C in 40% sterile glycerol. All bacteria were freshly grown at 37⁰C immediately prior to use under the following conditions. All strains were streaked to single colonies on tryptic soy broth (TSB) agar plates (BD BactoTM Tryptic Soy Broth (Sparks, MD). Liquid cultures were grown in TSB (BD Bacto TM Tryptic Soy Broth (Sparks, MD).

[0164] Staphylococcus epidermis strain RP62 was obtained from ATCC (ATCC35984). RP 62-8 is a stable phase variant of RP62 that does not test positive for PIA production. A Staphylococcus aureus strain is NCTC 8325.

EXAMPLE 2: Total Biofilm Assay (Staphylococcus epidermidis)

[0165] Aliquots of RP62 and RP62-8 cells were thawed at room temperature. The cells were then diluted into TSB media to an OD_OOO of 0.5 (1 cm pathlength). The cells were aliquoted into wells using the Thermo systems multidrop 384 instrument. 384 well plates (Costar TC treated plates (#3712) received 50μk 96 well (Costar TC treated (#3902) received 200μl aliquots. The plates were incubated statically for 20 hours at 37°C in a humidified chamber. After incubation the OD₆₂O of the plates was read with the Spectrafluor Plus to determine the effects on growth. Half the volume of the well of 0.1 mg/ml of Thioflavin T dye ((Sigma) dissolved in water) was added to the wells and incubated at room temperature for 10 minutes. The wells were decanted by inverting and gently shaking. The wells were next washed with sterile MiIIiQ water dispensed with the Multidrop 384 instrument. A single 70μl wash for 384 well plates and a 200 μl for 96 well plates was used. Following the wash the plates were decanted and dried on a paper towel. The wells were then read using the Spectrafluor Plus using the following settings. Fluorescent settings are: Excitation filter 430 nm, Emission filter 535 nm, Number of flashes — 5, Lag time 0 μs, integration time 40 μs, gain 70, bottom read. [0166] Figure 1 shows the emission profile of Thioflavin T in the presence and absence of S. epidermidis. Excitation is at 450 nm. Thioflavin T dye alone in water, RP62 cells alone in water and Thioflavin T in the presence of RP62 cells are as indicated. Thioflavin T in the presence of RP62 cells shows an emission maximum at approximately 495 nm.

EXAMPLE 3: Total Bio film Assay (Staphylococcus aureus)

[0167] An aliquot of selected S. aureus frozen stock cells is thawed at room temperature. The cells are then diluted into TSB media containing an additional 3% NaCl (w/v) to an OD^oo of 0.5 (1 cm pathlength). The addition of 3% NaCl has been shown to be critical for induction of PIA and biofilm formation for S. aureus under laboratory conditions. Alternatively, individual cells from overnight TSB agar plates at 37⁰C could also be used as innocula. [0168] The cells are aliquoted into wells using the Thermo systems Multidrop 384 instrument. 384 well plates (Costar TC treated plates (#3712)) received 50μl aliquots, 96 well (Costar TC treated (#3902) received 200μl aliquots. The plates are incubated statically for 16 to 20 hours at 37⁰C in a humidified chamber. Unlike S. epidermidis, where the biofilms are resilient and stable with time, all of the S. aureus strains tested to date become fragile with time so the exact timing for each strain varies with conditions. After incubation, the OD₆₂₀ of the plates is read with the Spectrafluor Plus to determine growth. Half the volume of the well of 0.1 mg/ml of Thioflavin T dye (Sigma), dissolved in water)) is added to the wells and incubated at room temperature for 10 minutes. The wells were decanted by inverting and gently shaking. For more fragile strains, the fluorescence can be read prior to washing. The OD_62O of the biofilm can also be read using the Spectrafluor plus. The wells are next washed with sterile MiIIiQ water dispensed with the Multidrop 384 instrument. A single 70 μl wash for 384 well plates and a 200 μl wash for 96 well plates. Following the wash the plates are decanted again and dried on a paper towel. The wells are then read using the Spectrafluor Plus using the following settings. For 96 well plates, to average out heterogeneity of biofilms, the option to read 4 points in the well is used (square pattern). The OD₆₂o of the adhered biomass is also determined, which is accurate for thicker films. The fluorescence settings are: Excitation filter: 430 nm, Emission filter: 535 nm, Number of flashes: 5, lag time: 0 μs, Integration time 40 μs, Gain 70, Bottom read.

[0169] Many strains of S. aureus do not adhere well to polystyrene or even TC treated polystyrene. Therefore, to improve adherence of the S. aureus cells to the plate wells, serum proteins are adhered to the wells. Calf serum proteins are diluted into carbonate buffer and added to the plates and allowed to adhere overnight at 4⁰C. The diluted serum proteins are decanted and washed once with sterile water prior to adding TSB and inoculating.

EXAMPLE 4: Silicone catheter shaking assay

[0170] Small sections of various medical device surfaces, including silicone and Teflon are added to a high innoculum of S. epidermidis cells. Incubation is carried out overnight at 4⁰C. The fragments are rinsed to remove non-adhered cells. The infected device fragments are next grown up in 16 mM glass test tubes with shaking in rich media to allow accumulation of biofilm on the device fragments. Planktonic bacterial cells seeded from the device fragments grow up quickly. Therefore, to allow nutrients to be used more predominately by the biofilm cells leading to increased accumulation, the biofilm coated device fragments are transferred to test tubes with fresh media at varied times throughout the assay. The biofilm cells are removed from the device fragments by soaking with detergent, vortexing and sonication. [0171] The quantitation of the biofilm on the device can be determined by viable cell counts and/or addition of Thioflavin-T. Quantitation using Thioflavin T can be carried out using fiber optic technology where the device delivers excitation radiation and detects surface fluorescence.

EXAMPLE 5: Screening for compounds that modulate biofilm formation. [0172] Screens are carried out using the total biofilm assay with S. epidermidis or S. aureus. The wells are TC treated or coated with serum proteins. The compounds, dissolved in DMSO are added to the wells prior to addition of cells. The final concentration of DMSO in the wells is no higher than 2%. The plates are incubated statically for 20 hours at 37⁰C in a humidified chamber. Following incubation, the OD₆₂₀ of the plates is read with the Spectrafluor Plus to determine effects on growth. Thioflavin-T is added and the cells are washed, decanted and read as in Example 2.

Other Embodiments

[0173] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the following claims.

[0174] All references cited herein are incorporated herein by reference in their entirety.

Claims

What is claimed is

1. A method for measuring biofilm formation by microorganisms comprising: a) providing a thioflavin, a thioflavin derivative, a styrylbenzene, a styrylbenzene derivative or a napthalene derivative to a sample; and b) measuring the fluorescence from the sample containing the compound to detect the presence of a biofilm or a microorganism capable of producing a biofilm.

2. The method of claim 1 wherein the compound is thioflavin or a thioflavin derivative.

3. The method of claim 2 wherein the thioflavin or thioflavin derivative is a compound of formula I

I or a salt thereof, wherein X is S, O or -CH;

Y is C or N, when Y is C the dashed line represents a bond, and when Y is N the dashed line is abesent;

R₁ is H or a lower alkyl group; R₂ is H, a lower alkyl group, -OH₅ -CO₂H, -CH₂CO₂H, -NH₂, -NHCH₃, 1, F, Cl, Br or

R₃ is H, I, F₃ Cl or Br; Rt is H, -CH₃ or absent;

|-N N-CH₃ R₅ is -NH₂, -NHCH₃, -N(CH₃)CH₃, -OH, -OCH₃ or \ — ' ; and

R₆ is H, I, F, Cl or Br.

4. The method of claim 2 wherein the thioflavin or thioflavin derivative is a benzathiazole of formula II

II or a salt thereof, wherein

R₇ is H₅ a lower alkyl group, -OH, -CO₂H, -CH₂CO₂H, I₅ F, Cl or Br; R.₈ is H, -CH₃ or absent; and

R₉ is -NH₂, -NHCH₃, -N(CH₃)CH₃, -OH, -OCH₃ or

5. The method of claim 2 wherein the thioflavin or thioflavin derivative is a benzathiazole of formula III

III or a salt thereof, wherein

Rio is H₅ a lower alkyl group, -OH₅ -CO₂H₅ -CH₂CO₂H₅ 1₅ F, Cl or Br;

R_n is H₅ F, Cl₅ Br₅ 1₅ -SO₃ ^', -SH₅ -NO₂, -NH₂ or a lower alkyl group;

Ri₂ is H₅ -CH₃ or absent; and

R,3 is -NH₂, -NHCH₃, -N(CH₃)CH₃, -OH or -OCH₃.

6. The method of claim 2 wherein the thioflavin or thioflavin derivative is a benzathiazole of formula IV

IV or a salt thereof, wherein

Ri₄ is H₅ a lower alkyl group, -OH, -CO₂H, -CH₂CO₂H₅ -NH₂, -NHCH₃, 1, F₅ Cl or Br and Ris is -NH₂, -NHCH₃, -N(CH₃)CH₃, -OH or -OCH₃.

7. The method of claim 2 wherein the compound is a benzoxazole of formula V

V or a salt thereof, wherein Ri6 is I, F, Cl or Br and Ri₇ and Ris are independently H, -CH₃ or -CH₂CH₃.

8. The method of claim 2 wherein the compound is an imadazopyridine of the formula

VI or a salt thereof, wherein

Ri9 is H, a lower alkyl group or I, Br₅ F or Cl and R20 and R₂i are independently H, -CH₃ or -CH₂CH₃.

9. The method of claim 2 wherein the compound is Thioflavin-T or Thioflavin-S.

10. The method of claim 1 wherein the compound is a styrylbenzene or a styrylbenzene derivative.

11. The method of claim 10 wherein the styrylbenzene or styrylbenzene derivative is a compound of formula VII

VII or a salt thereof, wherein R₂₂ is -OH or -0(Ci-C₄ alkyl); R₂₃ is -CO₂Me, -CO₂H or -OH; and R₂₄ is Br, I, F, Cl, H or -O(C_rC₄ alkyl).

12. The method of claim 11 wherein R₂₂ is -OH or -OCH₃,

R₂₃ is -CO₂H or -CO₂Me, and R₂₄ is Br.

13. The method of claim 11 wherein R₂₂ is -OH or -OCH₃,

R₂₃ is -CO₂H and

R₂₄ is Br, I₅ H or -OCH₃.

14. The method of claim 10 wherein the styrylbenzene or styrylbenzene derivative is a compound of formula VIII

VIII or a salt thereof, wherein R₂₅ is either I, Br or F.

15. The method of claim 1 wherein the compound is a napthalene derivative.

16. The method of claim 15 wherein the napthalene derivative is a compound of formula IX

R 26 Il ^27

IX or a salt thereof, wherein

R₂₆ is-OCH₃, -N(CH₃)CH₂CH₂F₃ -N(CH₃)CH₂CH₂I, -N(CH₃)CH₂CH₂Br, and

17. The method of claim 15 wherein the napthalene derivative is a compound of formula X

X or a salt thereof, wherein

R₂₈ is -OCH₃, -N(CH₃)CH₂CH₂F, -N(CH₃)CH₂CH₂I or -N(CH₃)CH₂CH₂Br and

R₂₉ is -C(CH₃)CO₂H, *Y O ^CH' or

.

18. The method of any of claims 1-17 further comprising providing a vessel to hold the sample.

19. The method of claim 18 wherein the vessel contains a plurality of projections to which biofϊlms can adhere.

20. The method of claim 18 wherein the vessel is coated with a biotic coating.

21. The method of claim 20 wherein the biotic coating is lysine, polylysine, serum proteins, mucin or similar compounds.

22. The method of claim 18 wherein the vessel includes a medical device or a portion thereof.

23. The method of claim 22 wherein the medical device is a catheter or a stent.

24. The method of any of claims 1-23 wherein the method is used to detect microorganisms or biofϊlms formed by microorganisms capable of producing a polysaccharide.

25. The method of claim 24 wherein the microorganisms include a Staphylococcus species.

26. The method of claim 25 wherein the Staphylococcus species is selected from the group consisting of, Staphylococcus epidermidis, Staphylococcus aureus, Staphylococcus haemolyticus, Staphylococcus saprophyticus, Staphylococcus hominis, Staphylococcus warneri, Staphylococcus cohnii, Staphylococcus capitis, Staphylococcus camosis and Staphylococcus lugdunensis or a combination thereof.

27. The method of claim 24 wherein the microorganisms include Escherichia coli.

28. The method of claim 24 wherein the microorganisms include Yersinia pestis.

29. The method of any of claims 1-28 further comprising a) providing a growth medium to a vessel; b) adding the sample to the vessel; c) incubating the sample in growth medium.

30. The method of any of claim 29 wherein the thioflavin, the thioflavin derivative, the styrylbenzene, the styrylbenzene derivative or the napthalene derivative is added after incubating the sample.

31. The method of claim 29 wherein the thioflavin, the thioflavin derivative, the styrylbenzene, the styrylbenzene derivative or the napthalene derivative is added at the beginning of incubating the sample.

32. The method of claim 29 wherein the thioflavin, the thioflavin derivative, the styrylbenzene, the styrylbenzene derivative or the napthalene derivative is added at any time from the beginning of incubating the sample to the end of the incubating the sample.

33. The method of any of claims 1-32 further comprising measuring fluorescence of the sample.

34. A method for measuring biofilm formation by a microorganism, wherein the method comprises a) providing a multiwell plate; b) providing a growth medium in a well in the plate; c) inoculating the well in the multiwell plate with a sample; d) incubating the inoculated medium for 2 to 24 hours; e) adding a thioflavin, a thioflavin derivative, a styrylbenzene, a styrylbenzene derivative or a napthalene derivative; and f) measuring the fluorescence.

35. The method of claim 34 wherein the multiwell plate is coated with a biotic coating.

36. The method of claim 35 wherein the biotic coating is lysine, poly Iy sine, serum proteins, mucin or similar compounds.

37. A method for identifying a compound that modulates a biofϊlm, the method comprising a) contacting sample containing microorganisms or a biofϊlm with a test compound; b) exposing the sample to a thioflavin, a thioflavin derivative, a styrylbenzene, a styrylbenzene derivative or a naphthalene derivative; and c) measuring the fluorescence from the sample.

38. The method of claim 37 wherein said microorganisms include Staphylococcus species.

39. The method of claim 45 wherein the Staphylococcus species is Staphylococcus selected from the following species, Staphylococcus epidermidis, Staphylococcus aureus, Staphylococcus haemolyticus, Staphylococcus saprophyticus, Staphylococcus hominis, Staphylococcus warneri, Staphylococcus cohnii, Staphylococcus capitis, Staphylococcus camosis, Staphylococcus lugdunensis or a combination thereof.

40. The method of claim 44 wherein the microorganisms include Escherichia coli or Yersinia pestis.

41. The method of claim 44 wherein the microorganisms include a microorganism or microorganisms that produce a polysaccharide structurally similar to the polysaccharide intercellular adhesion molecule of Staphylococcus epidermidis.

42. A kit for assaying a biofϊlm, the kit comprising: a) a vessel b) growth medium c) microorganisms selected from the group consisting of Staphylococcus species, Escherichia coli, or a microorganism that produces a polysaccharide or a combination thereof, and d) a thiofiavin, a thioflavin derivative, a styrylbenzene, a styrylbenzene derivative or a napthalene derivative or a combination thereof.

43. An in vivo method for detecting a biofilm or biofilm formation in a subject comprising a) administering a thioflavin, a thioflavin derivative, a styrylbenzene, a styrylbenzene derivative or a napthalene derivative to a subject, and b) measuring, imaging or detecting the compound in the subject.

44. The method of claim 43 wherein the compound is measured, imaged, or detected with gamma imaging, magnetic resonance imaging and/or magnetic resonance spectroscopy.

45. The method of claim 43 wherein the compound is measured, imaged, or detected with PET or SPECT.

46. The method of claim 43 wherein the subject is an animal or human patient suspected of having an infection caused by microorganisms.

47. The method of claim 46 wherein the subject is an animal or human patient who is immunocompromised.

48. A method of detecting biofilms in vitro, comprising: a) incubating a sample with a solution of a thioflavin, a Ihioflavin derivative, a styrylbenzene, a styrylbenzene derivative or a napthalene derivative; and b) measuring the fluorescence from the sample.

49. The method of claim 48 wherein the fluorescence is measured by a microscopic technique selected from the group consisting of bright-field, fluorescence, laser-confocal and cross-polarization microcopy.