GB2363842A - Micro-organism identification - Google Patents

Abstract

A method of identifying one or more micro-organisms in a fluid sample, said method comprising the steps of:- <SL> <LI>(i) optionally culturing the sample if necessary to increase the number of micro-organisms to a pre-determined range; <LI>(ii) applying an electric field across a portion of said fluid; <LI>(iii) measuring the velocity, displacement, zeta potential or electrophoretic mobility of any micro-organisms present following the application of said electric field; <LI>(iv) re-measuring the velocity, displacement, zeta potential or electrophoretic mobility of the fluid sample after incubation in the presence of a bioactive peptide; <LI>(v) comparing said measured velocities, displacements, zeta potentials or electrophoretic mobilities with tables of velocities, displacements, zeta potentials or electrophoretic mobilities of known micro-organisms measured under substantially identical experimental conditions to determine which, if any, micro-organisms are present. </SL>

Description

2363842 IDENTIFICATION METHOD

Field of the Invention

The present invention relates to a method and apparatus for the detection of the presence of specific micro-organisms in a fluid It is particularly applicable, but in no way limited, to identifying microbial pathogens.

Backqround to the Invention There are many applications in which it is important to be able to detect the presence of a specific micro-organism For example, in combating viral or bacterial infections, it is necessary to be able to identify the micro-organism responsible In certain military situations it is important to know quickly if there is an infective agent in the environment and if so, what it is In this context the term microorganism has a broad meaning It encompasses bacteria, viruses and fungi as well as an individual animal cell, for example a blood cell, or a plant cell, for example an alga.

In both these examples speed of analysis is extremely important For instance, in diagnosis of a medical problem the medical practitioner needs to know what organism is causing the symptoms within hours rather than days The most appropriate treatment can then be started straight away, giving the patient the best chance of a speedy recovery In some cases, such as meningitis, rapid and accurate diagnosis is a matter of life and death.

Under present arrangements samples are usually sent to a pathology laboratory for culture and subsequent identification By the very nature of the procedure this takes days rather than hours There may be more than one organism present which requires a number of different cultures in different media.

It is also known to detect micro-organisms by the use of specific probes that are designed to attach themselves to the micro-organism by covalent bonding and thus to attach a marker to them so that they may be detected by some physical property Unlike the present invention such techniques are slow and can only search for one micro-organism at a time.

The use of zeta potential measurements to identify micro-organisms has been proposed by Brown et al in GB 9907339 7 the entire text of which is hereby imported by reference Whilst this method is a major improvement over the prior art it does not always provide the sensitivity to differentiate uniquely between all micro- organisms Whilst it can usually be relied upon to provide valuable information on what class of organism is present it cannot always be used to identify which strain of organism is present.

Buggs and Green (J Bacteriol ( 1935) 30:447-451) examined the electrophoretic velocities of virulent and non-virulent strains of Corynebacterium diphtheriae They concluded that although there was a difference in electrophoretic velocity between toxigenic and non-toxigenic strains (the former is 19 8 % lower than the latter), the approach could not be used for determining the toxigenicity or non- toxigenicity of a given culture of Corynebacterium.

Dyar and Ordal (J Bacteriol ( 1946) 51:149-167) describe the effects of surface-active agents on the electrophoretic mobility of Micrococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Spirillum volutans, Bacillus pseudotetanicus, Mycobacterium smegmatis, M phlei, Chondrococcus columnaris and Spirochetes They studied the effect of the anionic surface-active agent 2- methyl-7-ethyl-undecanol-4-sulphate and the cationic agent cetyl pyridinium chloride on the mobility at 1, 10, 100 and 1000 p M For Spirillum volutans, Mycobacterium smegmatis, and M phlei, they also examined the effect of p H The aim of this work was to establish a method for the characterisation of bacterial surfaces.

Moyer (J Bacteriol ( 1936) 32:433-464) studied the electrophoretic mobility of Escherichia coli under various conditions No change in the mobility of the bacteria with p H could be noted between p H 4 and 7 when suspended in buffers of constant ionic strength.

Bayer and Sloyer (J Gen Microbiol ( 1990) 136:867-874) examined the electrophoretic mobility of a variety of Gram-negative and Gram-positive bacteria.

At neutral p H the magnitude of the electrophoretic mobility varied widely: the lowest value was for Cytophaga U 67 and the highest value was for organisms with polysaccharide capsules such as E coli K 1 They also report that ampicillin had no effect on the mobility, but polymyxin B and polymyxin B nonapeptide gave a reduction in mobility.

Lytle, Rice, Johnson and Fox (Appl Environ Microbiol ( 1999) 65:3222-3225)

disclose that p H and ionic strength have a differential effect on the electrophoretic mobility of E coli 0157:H 7 and wild type E coli.

Pelletier, Bouley, Cayuela, Bourlioux and Bellon-Fontaine (Appl Environ Microbiol ( 1997) 63:1725-1731) also show that the electrophoretic properties of 8 Lactobacillus strains varied with p H.

Ivanov, Fomchenkov, Khasanova and Gavryushkin (Microbiol ( 1997) 66:490495) report that the electrophoretic mobility of Pseudomanas fluorescens, E coli K- 12 and Mycobacterium phlei B-1291 treated with nickel, cadmium and lead ions changed in a p H range corresponding to the transition of bivalent metal ions to their monovalent hydroxylated forms Collins and Stotzky (Appl Environ Microbiol ( 1992) 58:1592-1600) also found that heavy metal chloride salts alters the electrokinetic properties of Bacillus subtilis, B megaterium, Pseudomanas aeruginosa and Agrobacterium radiobacter.

We have found that using phosphate buffer and varying the p H and varying other experimental conditions is sometimes not sufficient to discriminate between the four bacteria used in our experiments We have surprisingly found that phosphate buffer has a different effect on the change in velocity, displacement, zeta potential or electrophoretic mobility with p H than does a range of "Goods" buffers.

We have also surprisingly found that different buffer systems can have different effects on the variation of velocity, displacement, zeta potential or electrophoretic mobility with p H Thus one pair of buffer systems may produce a difference in velocity, displacement, zeta potential or electrophoretic mobility at a particular p H, whereas other combinations do not We have also found that different buffer systems at the same p H and ionic strength produce different values of velocity, displacement, zeta potential or electrophoretic mobility By choosing these more discriminatory buffer combinations, we have found that increasing the salt concentration (increasing the ionic strength) has a further discriminatory effect.

Electrophoretic mobility is the velocity a particle has per unit of electrical field strength, and typically has the units of pim per second per (Volt per cm) or 1 m/s/V Nlcm This value can either be measured under micro- electrophoresis conditions as described by Moyer (J Bacteriol ( 1936) 31:531-546) or by using a commercially available instrument, such as the Malvemrn Zetasizer 2000.

Zeta potential, C, is derived from electrophoretic mobility by the equation:

4 = Uni/e where u is the electrophoretic mobility, N is the viscosity and is the dielectric constant Van der Wal et al (Langmuir ( 1997) 13:165-171), however show that further factors need to be introduced into this equation to give a true conversion of electrophoretic mobility into zeta potential.

It will be appreciated that in solution the velocity and hence distance travelled by a micro-organism under the influence of an applied electrical field will be proportional to the electrophoretic mobility It follows therefore that it is not strictly necessary to compute the electrophoretic mobility or zeta potential in a series of experiments where the dielectric constant and viscosity of the various solutions are substantially constant It is therefore quite sufficient to determine the velocity or distance travelled per unit time providing, as stated above, the experimental conditions remain substantially constant This approach can simplify the computations significantly if image analysis is used.

Lysozyme (muramidase) is a relatively small ( 129 AA) secretory enzyme that catalyses the hydrolysis of specific kinds of polysaccharides comprising the cell walls of bacteria The effect of lysozyme is similar to the effect of penicillin which also weakens the cell walls of bacteria only by irreversibly inhibiting a transpeptidase enzyme required for cross linking peptidoglycan macromolecules formed in the biosynthesis of the cell wall Under normal conditions, bacteria grow very rapidly in some cases doubling in numbers more than once in an hour.

However, when cell wall cross linking is disrupted, bacteria tend to lyse in hypotonic media as a result of the mechanical weakening of their cell walls.

Lysozyme hydrolyzes preferentially the P-1,4 glucosidic linkages between N- acetylmuramic acid and N-acetylglucosamine which occur in the mucopeptide cell wall structure of certain micro-organisms, such as Micrococcus lysodeikticus A somewhat more limited activity is exhibited towards chitin oligomers Lysozyme hydrolyzes a number of structurally similar substrates but the best-known substrates for this enzyme are alternating polysaccharide copolymers of N-acetyl glucosamine (NAG) and N-acetyl muramic acid (NAM) which represent the "unit" polysaccharide structure of many bacterial cell walls Lysozyme cleaves at 1-1,4 glycosidic linkage, connecting the Cl carbon of NAM to the C 4 carbon of NAG The optimal substrate is a (NAG-NAM)3 hexasaccharide, with lysozyme cleaving at the NAM 4-O-NAG 5 glycosidic bond.

Ullman and Rubenstein in US Pat No 4,065,354 describe novel conjugated enzyme compositions for use in homogeneous immunoassays A wide variety of haptenic compounds, such as drugs of abuse, therapeutic drugs, naturally occurring hormones, and the like, are conjugated to the enzyme lysozyme for use as reagents in homogeneous enzyme immunoassays One or more of the haptens are conjugated by relatively short chains or linking groups to lysozyme to provide a product retaining a substantial proportion of the original enzyme activity, which is greatly reduced when the conjugated hapten is bound to antibody.

Pugia in US Pat No 5,753,455 discloses the determination of lysozyme in urine by contacting the urine with a reagent system containing a buffer and a protein error indicator dye The analysis is carried out by contacting the fluid suspected of containing lysozyme with a test reagent comprising a protein error indicator dye, which undergoes a detectable colour change when contacted with protein in a buffered solution He discloses a method which comprises adding to the test reagent an alkyl sulfonic acid having from 9 to 15 carbon atoms or a salt of said sulfonic acid wherein the size of the alkyl group and the concentration of alkyl sulfonic acid in the aqueous test sample are such that the detectable colour change is caused by lysozyme in the test sample but a detectable colour change is not caused by human serum albumin and/or Ig G or other urinary protein present in the sample.

In US Pat No 5,994,067, Wood and Sydiskis describe a rapid method for detection of bacteria The method involves treating a sample suspected of containing bacteria with a bactericidal peptide and a fluorescent marker for bacterial DNA The preferred bactericidal peptide is a cecropin, which does not "burst" the bacterium's membranes but instead forms a pore or hole through which the fluorescent indicator can enter, much the same as calcium ion channels If bacteria are present in a sample, there will be an increase in fluorescence.

Cecropins are small basic peptides, 30-40 amino acids in length, and have been shown to have antibacterial properties They have been isolated from insects and the intestines of pigs Cecropins have marked antibacterial activities against a variety of bacteria tested including a variety of pathogenic gram- positive and gram- negative bacteria, fungi, protozoa and enveloped viruses Cecropin B, isolated from insects, is active on wild-type enteric bacteria and their lipopolysaccharide/lipid A mutants that have defective outer membranes, as exemplified by K-12 strains of E.

coli The mode of action of the cecropin peptides involves pore formation at the cytoplasmic membrane, and their action resembles the activity of quaternary detergents.

The property of certain peptides to induce lysis of procaryotic microorganisms such as bacteria is known For example, U S Pat Nos 4,355,104 and 4,520,016 to Hultmark et al describe the bacteriolytic properties of some cecropins against Gram-negative bacteria Quite interestingly, the cecropins described in the Hultmark et al patents were not universally effective against all Gram- negative bacteria Other lytic peptides heretofore known include, for example, the sarcotoxins and lepidopterans Such peptides generally occur naturally in the immune system of Sarcophaga peregrina and the silkworm, lepidopteran, respectively, as reported in Nakajima et al, The Journal of Biological Chemistry, vol 262, pp 16651669 ( 1987) and Nakai et al, Chem Abst 106:214351 W ( 1987).

In US Pat No 5,861,478, Jaynes discloses synthetic lytic peptides, particularly designed and constructed to encompass those structural properties believed to be associated with lytic and proliferative function: aligned amphipathic x- helical conformation, with positive charge density These synthetic lytic peptides are designed to function in the treatment of plants and animals against microbial infections including bacterial, yeast, fungal, viral and protozoan infections.

In US Pat No 5,889,148, Lee et al disclose novel antibiotic peptides which possess antibacterial and/or antifungal activities They showed that a number of chemically-synthesized peptides which are derived from Tenecin, show superior antibacterial and/or antifungal activities.

It has been found that many organisms synthesize proteins (or peptides) which are degraded to relatively small hydrophobic or amphipathic, bioactive peptides These peptides exhibit antibiotic, fungicidal, virucidal, hemolytic and/or tumoricidal activities by interacting with membranes and forming transmembrane channels that allow the free flow of electrolytes, metabolites and water across the phospholipid bilayers Most of these peptides appear to function in biological warfare There are many designations given to these bioactive peptides They include the magainins, cecropins, melittins, defensins, bacteriocidins, etc The proteins in each family within this functional superfamily are homologous, but they exhibit little or no significant sequence similarity with members of the other families.

Thus, each family may have evolved independently However, certain common structural features observed between members of distinct families suggest that at least some of these families share a common ancestry.

The generalised transport reaction catalysed by channel-forming amphipathic peptides is:

small solutes, electrolytes and water (in) small solutes, electrolytes and water (out).

Several families of eukaryotic CA Ps, each from a different group of organisms, are recognised.

Cecropins are produced by insects, particularly under conditions of infection.

Cecropins A, B and D are close homologues consisting of 35 39 residues They are found in the pupae of the cecropin moth, but related homologues named lepodopteran, bactericidin, moricin and sarcotoxin are produced by other insects.

The following table gives a summary of some of these For the purpose of this disclosure these related homologues are defined as cecropin-like proteins.

Name Source(s) Example Cecropin A, B and C Insects Cecropin A, B and C precursor precursor of Hylaphora cecropia ( 64 aas; sp P 01507) Hyphancin III Insects Hyphancin III E precursor E precursor of Hyphantria cunea ( 63 aas;sp P 50720) Moricin precursor Insects Moricin precursor of Bombyx mori ( 59 aas; sp P 48821) Bactericidin B-5 P Insects Bactericidin B-5 P precursor precursor of Manduca sexta ( 61 aas; sp P 14665) Sarcotoxin IA precursor Insects Sarcotoxin IA precursor of Sarcophaga peregrina ( 63 aas; sp P 08375) Lysosyme and related enzymes and the antibacterial polypeptides described above share a common property As bioactive peptides they are able to bring about specific chemical changes in the cell membranes surrounding certain micro-

organisms These chemical changes can be distinguished from the physicochemical changes which are brought about by altering the p H or ionic strength of the medium containing the micro-organism.

It has unexpectedly been discovered that bioactive peptides that cause a change to a group or groups on a micro-organism surface can be used in a method of detecting and identifying specific micro-organisms involving the measurement of electrophoretic mobility or related physical properties.

Summary of the Invention

According to a first aspect of the present invention there is provided a method of identifying one or more micro-organisms in a fluid sample, said method comprising the steps of:- (i) optionally culturing the sample if necessary to increase the number of micro- organisms to a pre-determined range; (ii) applying an electric field across a portion of said fluid; (iii) measuring the velocity, displacement, zeta potential or electrophoretic mobility of any micro-organisms present following the application of said electric field; (iv) re-measuring the velocity, displacement, zeta potential or electrophoretic mobility of the fluid sample after incubation in the presence of a bioactive peptide; (v) comparing said measured velocities, displacements, zeta potentials or electrophoretic mobilities with tables of velocities, displacements, zeta potentials or electrophoretic mobilities of known micro-organisms measured under substantially identical experimental conditions to determine which, if any, micro-organisms are present.

This method provides improved sensitivity and selectivity over prior art methods because the bioactive peptide exerts an effect on the microorganism which manifests itself as a change in the measured parameters In this context a bioactive peptide is one which produces some change, either permanent or reversible, to a micro-organism.

Preferably the bioactive peptide is selected from the group consisting of lysozyme, mutanolysin, lyticase, chitinase, mucopeptide amidohydrolase, N-

acetylglucosamine deacetylase,,8 ( 1-6) and /( 1-3) glycanases, proteases, mannase, cecropins and cecropin-like proteins, Magainin I and II, amide, Cecropin P 1, A and B, Defensin HNP-1 and HNP-2, Nisin, Lactoferricin, Buforin II, indolocidin, ranalexin, sarcotoxins, lepidopterans, moricins and hyphancins.

In a particularly preferred embodiment the bioactive peptide is lysozyme.

In a further preferred embodiment the bioactive peptide is mutanolysin.

In a further preferred embodiment the bioactive peptide is cecropin or a cecropin-like peptide.

In a still further preferred embodiment the bioactive peptide is magainin.

In a particularly preferred embodiment aliquots of the fluid sample produced at the end of optional stage (i) are incubated with or suspended in an array of different bioactive peptides, including at least one control without bioactive peptide present.

By using an array of experimental conditions which include an untreated control a number of readings can be generated very rapidly This provides a unique fingerprint which enables a micro-organism to be identified particularly rapidly and accurately.

Preferably in optional step (i) the sample is cultured in a medium selective for a particular organism or class of organisms, said modification providing a method of distinguishing between micro-organisms that otherwise have similar velocities, displacements, zeta potentials or electrophoretic mobilities.

In a preferred embodiment the comparison between the measured velocity displacement, zeta potential or electrophoretic mobility and said table of known velocities, displacements, zeta potentials or electrophoretic mobilities is carried out using a computer program, the result being displayed on a computer screen or by way of a printout Information may also be provided on preferred treatment(s) This could be by way of a list of suitable antibiotics with indications and contraindications.

Preferably said fluid sample is obtained from a human or animal body and said method provides an indication of the cause of an infection in said human or animal body.

In a further preferred embodiment aliquots of the fluid sample containing the micro-organism(s) are, in addition to incubation with one or more bioactive peptide, suspended in a plurality of different solutions, said solutions representing different experimental conditions differing in one or more of the parameters selected from the group of parameters consisting of p H, ionic strength, concentration of surface-active agent, concentration of heavy metal ions, concentration of chelating or complexing agent, concentration of organic solvent, concentration of dye or stain, concentration of polyol, or concentration of polyether; and subsequently comparing said measured velocities, displacements, zeta potentials or electrophoretic mobilities with tables of velocities, displacements, zeta potentials or electrophoretic mobilities of known micro-organisms measured under substantially identical experimental conditions to determine which, if any, micro-organisms are present.

These additional experimental conditions enable micro-organisms with otherwise very similar electrophoretic mobility to be distinguished one from another.

According to a second aspect of the present invention there is provided an apparatus for carrying out the methods described herein, said apparatus comprising:- (i) means for applying an electric field across a measurement cell; (ii) a light source for illuminating the measurement cell; (iii) detecting means for detecting light scattered by micro-organisms present in the cell; (iv) means for analysing the scattered light to provide a measurement of the speed of movement of a micro-organism(s); (v) means for computing the velocity, displacement, zeta potential or electrophoretic mobility of said micro-organism(s); (vi) means for comparing said measured velocities, displacements zeta potentials or electrophoretic mobilities, with the velocities, displacements, zeta potentials or electrophoretic mobilities of known organisms; characterised in that the apparatus further comprises an array of measurement cells containing one or more bioactive peptides selected from the group comprising lysozyme, mutanolysin, lyticase, chitinase, mucopeptide amidohydrolase, N-

acetylglucosamine deacetylase,,?( 1-6) and,8 ( 1-3) glycanases, proteases, mannase, cecropins and cecropin-like proteins, Magainin I and II, amide, Cecropin P 1, A and B, Defensin HNP-1 and HNP-2, Nisin, Lactoferricin, Buforin II, indolocidin, ranalexin, sarcotoxins, lepidopterans, moricins and hyphancins.

Preferably the array of measurement cells contains, in addition to one or more bioactive proteins, a plurality of different solutions, said solutions representing different experimental conditions differing in one or more of the parameters selected from the group of parameters consisting of p H, ionic strength, concentration of surface-active agent, concentration of heavy metal ions, concentration of chelating or complexing agent, concentration of organic solvent, concentration of dye or stain, concentration of polyol, or concentration of polyethers.

The present invention also provides a method for measuring the velocity of micro-organisms in solution following the application of an electric field, by means of image analysis According to this aspect of the invention, a first image of part of the fluid in which the micro-organisms are suspended is obtained prior to the application of the electric field to a portion of the fluid and a second image is obtained after the electric field has been applied for a predetermined period of time The displacement of the micro-organisms is computed from the two images, from which the velocity of the micro-organisms may be obtained This process is optionally repeated a number of times.

Image analysis may be performed by digital analysis of the images using a computer program, or it may be performed by optical information processing which exploits the large information capacity and parallel processing capability of optical systems to perform tasks such as pattern recognition This can involve the use of diffractive optical elements to process optical fields both spatially and temporally.

Detailed Description of the Preferred Embodiments

The present invention is a method for determining a characteristic fingerprint for a micro-organism The micro-organism may be a bacterium, a fungus, a virus or it may be an individual animal cell, for example a blood cell, or a plant cell, for example an alga.

The present invention is also a method for detecting the presence of a micro- organism in a sample The micro-organism may be any micro-organism that is susceptible to an enzyme composition The micro-organism is preferably a bacterium.

The method comprises measuring the velocity, displacement, zeta potential or electrophoretic mobility of particles present in a sample suspected of containing the micro-organism The sample is then contacted with a composition comprising one or more bioactive proteins for a predetermined time The velocity, displacement, zeta potential or electrophoretic mobility is again measured, and a change in the electrophoretic mobility of any of the particles present in the sample is indicative of the presence of a micro-organism.

In the description which follows the term "zeta potential or electrophoretic mobility" is intended to include the physical properties velocity and displacement as appropriate.

The sample containing the micro-organism may be derived from a number of sources For example, the sample may be a sample obtained from a human or animal subject: the sample may be of urine or blood, or it may be derived from swabbing, for example swabbing the throat, or from faeces The sample may also be obtained from a water supply The sample may be a food material, or it may be obtained from a food material by swabbing the surface of the food and transferring the swab to a liquid to cause any micro-organism present to be transferred from the surface of the food to the solution The sample may also be obtained from a food material by homogenising the food to cause any micro-organism present to be transferred from the food to the homogenising solution The sample may be obtained from the atmosphere by drawing air through a liquid to cause any micro- organisms present in the atmosphere to be transferred to the liquid Prior to measurement, the sample containing the micro-organism may be contacted with nutrients and incubated at a suitable temperature to cause the micro- organisms in the sample to grow and divide In one embodiment, the nutrients added and the temperature used are chosen such that only one or only one group of micro-

organisms are caused to grow and divide.

The bioactive peptide may be an enzyme and the enzyme may be any enzyme that catalyses a change to a group or groups on the micro-organism surface These groups include: murein (or peptidoglycan, mucopeptide), teichoic acid, lipopolysaccharide, lipoprotein, phospholipid, polysaccharide and protein.

Whilst not wishing to be bound to a particular theory, it is believed that a change to these groups alters the electrical surface properties of the micro- organism leading to a change in the measured zeta potential or electrophoretic mobility Enzymes that may be used in this invention include but are not limited to: lysozyme, mutanolysin, lyticase, chitinase, mucopeptide amidohydrolase, N-acetylglucosamine deacetylase, p( 1-6) and P( 1-3) glycanases, proteases, and mannase Preferred enzymes are lysozyme and mutanolysin An example of the use of lysozyme is given in Example 12 and an example of mutanolysin is given in Example 13.

This list is not intended to be exhaustive or limiting It is intended thatthis disclosure should encompass any bioactive peptide enzyme, known or yet to be discovered, which is capable of causing a change to a group or groups on the surface of a micro-organism EDTA or other chelating agent may be used in combination with any of these enzymes to destabilize the outer membrane of Gram negative cells, making the peptidoglycan layer accessible to the enzyme used.

The bioactive peptide may also be any antibacterial peptide that binds to the micro-organism surface and creates a pore Whilst not wishing to be bound to a particular theory, it is believed that this binding process changes the electrical surface properties of the micro-organism leading to a change in the measured zeta potential or electrophoretic mobility Antibacterial peptides that may be used in this invention include: Magainin I and II; Magainin II amide; Cecropin P 1, A and B; Defensin HNP-1 and HNP-2; Nisin; Lactoferricin; Buforin II; indolocidin; and ranalexin Cecropin, cecropin-like proteins and magainin are particularly preferred antibacterial peptides.

The invention also provides a method for distinguishing a micro-organism in a mixture containing eukaryotic cells, or cells which are not susceptible to the enzyme composition This embodiment comprises measuring the zeta potential or electrophoretic mobility of particles present in a sample suspected of containing the micro-organism in the presence of other cells The sample is then contacted with a composition comprising an enzyme The zeta potential or electrophoretic mobility is again measured, and a change in the electrophoretic mobility of any of the particles present in the sample is indicative of the presence of a micro-organism This embodiment is particularly useful for detecting a micro-organism in a specimen taken from a human or animal subject that also contains cells from the subject Such samples include urine, faecal, or blood samples; throat, wound or genital swabs; and the like.

The present invention further provides a method for determining a characteristic fingerprint for a micro-organism The fingerprint comprises zeta potential values or electrophoretic mobility values obtained in the presence of an enzyme composition and under one or more different conditions.

Optionally in a further embodiment the composition of the solution is chosen such that it comprises a predetermined buffer system at a predetermined concentration Particularly preferred buffers include the following:

Buffer Abbreviation N-(Carbamoyl-methyl)-2-amino-ethane-sulfonic acid ACES N-( 2-Acetamido)-2-iminodiacetic acid ADA 2-amino-2-methyl-1,3-propanediol 2-amino-2-methyl-1 -propanol 3-amino-l-propanesulfonic acid 3-(l 1,1-Dimethyl-2-hydroxy-ethyllamino)-2-hydroxy-propane-sulfonic AMPSO acid N,N-bis( 2-Hydroxyethyl)-2-aminoethane-sulfonic acid BES N,N-bis( 2-Hydroxyethyl)-glycine BICINE bis( 2-Hydroxyethyl)imino-tris(hydroxymethyl)-methane BIS-TRIS 1,3-bis(trislHydroxy-methyll-methylamino)-propane BIS-TRIS PROPANE 4-(Cyclohexylamino)-1-butanesulfonic acid CABS 3-(Cyclo-hexyl-amino)-1-propane-sulfonic acid CAPS 3-(Cyclohexylamino)-2-hydroxy-1-propane-sulfonic acid CAPSO 2-(N-Cyclo-hexyl-amino)-ethane-sulfonic acid CHES 3-(N,N-bisl 2-Hydroxyethyll-amino)-2-hydroxy-propanesulfonic acid DIPSO N-( 2-Hydroxyethyl)-piperazine-N'-( 3-propane-sulfonic acid) EPPS Ethanolamine Glycine N-( 2-Hydroxyethyl)piperazine-N'-( 4-butanesulfonic acid) HEPBS N-( 2-Hydroxy-ethyl)piper-azine-N'-( 2-ethane-sulfonic acid) HEPES N-( 2-Hydroxyethyl)piper-azine-N'-( 2-hydroxy-propanesulfonic acid) HEPPSO Imidazole 2-(N-Morpholino)ethane-sulfonic acid MES 4-(N-Morpholino)butanesulfonic acid MOBS Buffer Abbreviation 3-(N-Morpholino)propane-sulfonic acid MOPS 3-(N-Morpholino)-2-hydroxy-propanesulfonic acid MOPSO Piperazine-N,N'-bis-( 2-ethanesulfonic acid) PIPES Piperazine-N,N'-bis-( 2-hydroxy-propane-sulfonic acid) POPSO N-tris(Hydroxy-methyl)-methyl-4-amino-butane-sulfonic acid TABS N-tris(Hydroxy-methyl)methyl-3-amino-propane-sulfonic acid TAPS 3-(N-trislHydroxymethyil-methyl-amino)-2-hydroxy-propane-sulfonic TAPSO acid N-tris(Hydroxymethyl)-methyl-2-aminoethane-sulfonic acid TES N-tris(Hydroxy-methyl)methyl-glycine TRICINE Triethanolamine Tris(hydroxy-methyl)amino-methane Tris The above list of buffers is not intended to be either exhaustive or limiting It is intended that this disclosure should cover any buffer or buffer system, known or yet to be discovered.

In another embodiment the composition of the solution is chosen such that it has a predetermined value of ionic strength Preferably the ionic strength is less than 0 05 so that the current passing through the sample is less than 20 m A The ionic strength can be varied by adding an ionic inorganic salt such as sodium chloride.

In a further embodiment the composition of the solution is chosen such that it contains a predetermined concentration of a surface-active agent A surface-active agent may be a non-ionic detergent, an anionic detergent, a cationic detergent or a zwitterionic detergent Examples of surface-active agents include the following:

Anionic detergents: Zwitterionic detergents:

Aerosol 22 CHAPS { 3-(l 3-Cholamidopropyll- Aerosol-OT dimethylammonio)-1 -propane- Salts of: sulfonate)} Alginic acid CHAPSO { 3-(l 3-Cholamidopropyll- Caprylic acid dimethylammonio)-2-hydroxy-1- Cholic acid propane-sulfonate} Anionic detergents: Zwitterionic detergents:

1-Decanesulphonic acid N-Decyl-N,N-dimethyl-3-ammonio-1- Dehydrocholic acid propanesulphonate Deoxycholic acid N-Hexadecyl-N,N-dimethyl-3Dioctyl sulphosuccinate ammonio-1-propanesulphonate 1-Dodecanesulphonic acid N-Octadecyl-N,N-dimethyl-3- Glycocholic acid ammonio-1-propanesulphonate Glycodeoxycholic acid N-Octyl-N,N-dimethyl-3-ammonio-1- 1-Heptanesulphonic acid propanesulphonate 1-Hexanesulphonic acid N-Tetradecyl-N,N-dimethyl-3N-lauroylsarcosine ammonio-1 -propanesulphonate Lauryl sulphate Phosphatidylcholine 1-Nonanesulphonic acid 1-Octanesulphonic acid 1-Pentanesulphonic acid Taurocholic acid Taurodeoxycholic acid Niaproof Cationic Detergents Nonionic detergents Alkyltrimethylammonium bromides: BIGCHAP {N,N-bis( 3-D-Glucon- Dodecyltrimethylammonium bromide amidopropyl)-cholamide} Hexadecyltrimethylammonium bromide Decanoyl-N-methylglucamide Tetradecyltrimethylammonium bromide n-Decyl-a-D-glucopyranoside Benzalkonium chloride n-Decyl-p-D-glucopyranoside Benzethonium chloride n-Decyl-p-D-maltopyranoside Benzyldimethyldodecylammonium bromide Deoxy-BIGCHAP {N,N-bis( 3-Glucon- Benzyldimethylhexadecylammonium bromide amido-propyl)-deoxycholamide} Benzyltrimethylammonium methoxide n-Dodecyl-P-D-glucopyranoside Cetyldimethyldiethylammonium bromide n-Dodecyl-a-D-maltoside Cetylpyridinium chloride n-Dodecyl-p-D-maltoside Decamethonium bromide Heptanoyl-N-methylglucamide Dimethyldioctadecylammonium bromide n Hepty Dgucopyranosde n-Heptyl-13-D-glucopyranoside Methylbenzethonium chloride Methyl mixed trialkylammonium chloride n-Heptyl D-hglucopyranoside Methyltrioctylammonium chloride n-Hexyl D-glucopyranoside N,N',N'-polyoxyethylene( 10)-N-tallow-1,3 Igepal CA-360 diaminopropane 1-Monooleoyl-rac-glycerol Nonanoyl-N-methylglucamide n-Nonyl-a-D-glucopyranoside n-Nonyl-P-D-glucopyranoside Octanoyl-N-methylglucamide n-Octyl-a-D-glucopyranoside n-Octyl-P-D-glucopyranoside Octyl-3 D-thiogalactopyranoside Octyl-P-D-thioglucopyranoside Polyoxyethylene esters Polyoxyethylene ethers Polyoxyethylenesorbitan esters Sorbitan esters Tergitol n-Tetradecyl-P-D-maltoside Tritons Tyloxapol n-Undecyl-O-D-glucopyranoside The above list of surface-active agents is not intended to be either exhaustive or limiting It is intended that this disclosure should cover any surface- active agent known or yet to be discovered.

In a further embodiment the composition of the solution is chosen such that it contains a predetermined concentration of a dye or stain of the type used for staining or dyeing micro-organisms The stain may be: acid fuschin, alcian blue 8 GX, alizarin red S, Auramine 0, Azocarmine G, Azure A, Azure B, Bismarck brown Y, brilliant cresyl blue aid, brilliant green, carmine, congo red, cresyl violet acetate, crystal violet, eosin B, eosin Y, erythrosine B, fast green FCF, giemsa stain, hematoxylin, indigo carmine, janus green B, Jenner's stain, light green SF yellowish, malachite green oxalate, methyl blue, methylene blue, methyl green, methyl violet 2 B, neutral red, Nile blue A, orange II, Orange G orcein, pararosaniline chloride, phloxine B, pyronin B, pyronin Y, rose Bengal, safranin 0, Sudan II Il, Sudan IV, Sudan black B, toluidine blue O or Wright stain.

It will be appreciated that these are just examples of stains which might be used advantageously in this method However, this list is not intended to be limiting in any way It is intended that this disclosure should cover any stains, known or yet to be discovered, which cause coloration of a micro-organism.

In a further embodiment the composition of the solution is chosen such that it contains a predetermined concentration of an organic solvent The solvent may be a water-miscible solvent such as an alcohol, acetonitrile, DMSO, or THF The solvent may be a water-immiscible solvent, such as toluene, ethyl acetate, or ether used at a concentration at or below its maximum solubility in water so that a single aqueous phase is obtained Once again, a wide variety of organic solvents can be used in this method and the examples given above are by way of illustration only.

In a further embodiment the composition of the solution is chosen such that it contains a predetermined concentration of a heavy metal salt Suitable heavy metal salts are salts of copper, mercury, lead, calcium, chromate and the like.

In a further embodiment the composition of the solution is chosen such that it contains a predetermined concentration of a chelating agent The chelating agents may be ethylene diamine, DTPA (diethylenetriaminepenta-acetic acid), EDTA (ethylenediamine tetra-acetic acid), EGTA (ethylene glycol-bis(p 3- aminoethyl ether) N,N,N',N'-tetra-acetic acid), Dimercaprol ( 2,3-dimercapto-1-propanol), HEDTA (N- A 4 hydroxyethylethylenediaminetriacetic acid), citric acid, gluconates, or NTA (nitrilotri- acetic acid).

In a further embodiment the composition of the solution is chosen such that it contains a predetermined concentration of a polyol or polyether Suitable polyethers include polyethylene glycol and polyproylene glycol Polyols include monosaccharides, disaccharides, polysaccharides and carbohydrates in general.

In a preferred embodiment, the sample containing the micro-organism is mixed with two or more different solutions and the electrophoretic mobility of the micro-organism in each of the different solutions is measured The electrophoretic mobility data obtained under these defined but differing conditions provide a fingerprint characteristic of the micro-organism In an optional further step, the electrophoretic mobility data may be transformed to zeta potential data.

In another embodiment, the sample containing the micro-organism is mixed with a solution of predetermined composition to form a first mixture and the electrophoretic mobility of the micro-organism in the first mixture is measured At least one more different solution is mixed with this first mixture to form second and subsequent mixtures, and the electrophoretic mobility of the micro- organism in each of these subsequent mixtures is measured The electrophoretic mobility data obtained under these defined but differing conditions provide a fingerprint characteristic of the micro-organism In an optional further step, the electrophoretic mobility data may be transformed to zeta potential data.

In another preferred embodiment, a sample containing one or more unknown micro-organisms is obtained Aliquots of the sample are then mixed with two or more different solutions and the electrophoretic mobility of the microorganism or micro-organisms in each of the different solutions is measured In an optional further step, the electrophoretic mobility data may be transformed to zeta potential data The electrophoretic mobility and/or zeta potential data obtained under these defined but differing conditions is compared to the fingerprints of known micro- organisms to determine the identity of the one or more micro-organisms present in the sample In an optional further step the database will provide further information about the micro-organism or micro-organisms present For example in a clinical or veterinary application, the database may provide therapeutic information relating to the treatment of any disease associated with the presence of the micro- organism.

In another embodiment, the sample containing one or more unknown microorganisms is mixed with a solution of predetermined composition to form a first mixture and the velocity, displacement, zeta potential or electrophoretic mobility measured At least one more different solution is mixed with this first mixture to form second and subsequent mixtures, and the velocity, displacement, zeta potential or electrophoretic mobility of the micro-organism in each of these subsequent mixtures is measured In an optional further step, the electrophoretic mobility data may be transformed to zeta potential data The velocity, displacement, zeta potential or electrophoretic mobility data obtained under these defined but differing conditions is compared to the fingerprints to determine the identity of the one or more micro-organisms present in the sample In an optional further step the database will provide further information about the micro-organism or micro- organisms present For example in a clinical or veterinary application, the database may provide therapeutic information relating to the treatment of any disease associated with the presence of the micro-organism.

In an optional further step the database will provide further information about the micro-organism or micro-organisms present For example in a clinical or veterinary application, the database may provide therapeutic information relating to the treatment of any disease associated with the presence of the micro- organism.

The number of different conditions used will increase the discriminatory power of the fingerprint obtained Preferably between 2 and 20 different conditions are applied Most preferably between 4 and 12 different conditions are applied.

The electrophoretic mobility may be measured using any technique known to the art For example the micro-electrophoretic technique of Moyer (J Bacteriol ( 1936) 31:531-546) may be used, or a commercially available instrument such as the Malvern Zetasizer 2000 may be utilised Methods of measurement are also described in GB 99073397, incorporated herein by reference.

It will be appreciated that this method depends on having a database of measured values of velocity, displacement, zeta potential or electrophoretic mobility for known micro-organisms obtained under a wide variety of known but different experimental conditions These measurements, taken over a wide range of experimental conditions, provide unique fingerprints of each micro- organism The wide variety of experimental conditions permit micro-organisms with otherwise similar electrophoretic mobilities to be distinguished.

The sample containing the micro-organism may be derived from a number of sources To provide the database of fingerprints, known micro-organisms are cultured to provide the sample Where the method is to be applied to the determination of micro-organisms, the sample may be from any source for which the identity of any micro-organisms present in the sample is required For example, the sample may be a sample obtained and removed from a human or animal subject:

the sample may be of urine or blood, or it may be derived from swabbing the throat, or from faeces The sample may also be obtained from a water supply The sample may be a food material, or it may be obtained from a food material by swabbing the surface of the food and transferring the swab to a liquid to cause any micro- organism present to be transferred from the surface of the food to the solution The sample may also be obtained from a food material by homogenising the food to cause any micro-organism present to be transferred from the food to the homogenising solution The sample may be obtained from the atmosphere by drawing air through a liquid to cause any micro-organisms present in the atmosphere to be transferred to the liquid Prior to measurement, the sample containing the micro-organism may be contacted with nutrients and incubated at a suitable temperature to cause the micro-organisms in the sample to grow and divide In one embodiment, the nutrients added and the temperature used are chosen such that only one or only one group of micro-organisms are caused to grow and divide.

As an alternative to the measurement of electrophoretic mobility, image analysis techniques can be used to determine the velocity/distance travelled by a micro-organism under standard conditions In the context of the present invention the optimum physical property to be measured may be electrophoretic mobility, zeta potential, displacement or velocity and the optimum choice will be made after experimentation Where the term electrophoretic mobility is used it is intended to encompass any of the foregoing physical properties.

In one embodiment where velocity is measured, a first image of part of the fluid in which the micro-organisms are suspended is obtained prior to the application of the electric field to a portion of the fluid, and a second image is obtained after the electric field has been applied for a predetermined period of time The change in displacement of the micro-organisms is computed from the two images, from which the velocity of the micro-organisms may be obtained This process is optionally repeated a number of times Image analysis may be performed by digital analysis of the images using a computer program, or it may be performed by optical information processing which exploits the large information capacity and parallel processing capability of optical systems to perform tasks such as pattern recognition This can involve the use of diffractive optical elements to process optical fields both spatially and temporally.

This disclosure is intended to encompass any known method of image analysis which is capable of providing the level of accuracy required for these measurements It is also intended to encompass image analysis techniques yet to be discovered.

Example 1 Effect of buffer, p H and ionic strength The bacterial strains used in this study were Escherichia coli W 3110, Bacillus cereus, Enterococcus faecalis and Pseudomonas aeruginosa Cultures were grown in nutrient broth at 37 C with shaking until the optical density at 600 nm was 0 3 The buffers used and their respective conductivities are shown in Table 1.

Table 1

Buffers chosen for the measurement of zeta potentials of the initial test bacterial species (all buffers used at 5 m M) Buffer p H Conductivity (m S) Citrate 3 3 0 31 Glycine 3 4 0 29 Acetate 4 1 0 10 Glycine 4 3 0 06 Succinate 5 2 0 34 Imidazole 5 8 0 37 MES 6 0 0 09 Imidazole 6 8 0 40 Phosphate 7 2 0 43 Tris 7 8 0 26 Tricine 7 9 0 12 Borate 8 7 0 54 Diethanolamine 8 8 0 23 Trimethylamine 9 5 0 30 Glycine 9 7 0 19 Phosphate 10 1 O 68 Triethylamine 10 3 0 22 The buffer solution was filtered through a 0 2 gm filter prior to use to remove small particles that may interfere with subsequent electrophoretic measurements.

Electrophoretic mobilities and the derived zeta potentials were obtained by analysing the solutions in a Malvern Zetasizer 2000 Figure 1 shows the variation of zeta potential with conductivity and shows how Pseudomonas aeruginosa and Bacillus cereus, may be distinguished from Enterococcus faecalis and Escherichia coli.

Example 2 Effect of organic solvents The bacterial strains used in this study were Escherichia coli W 3110, Bacillus cereus, Enterococcus faecalis and Pseudomonas aeruginosa Cultures were grown in nutrient broth at 370 C with shaking until the optical density at 600 nm was O 3.

An aliquot of each culture ( 1001 i) was added to 10 ml of 5 m M MES buffer, p H 6 0, containing 5 % sucrose and either 0, 3, 10 or 30 % methanol The buffer solution had been filtered through a 0 2 plm filter prior to use to remove small particles that may interfere with subsequent electrophoretic measurements.

Electrophoretic mobilities and the derived zeta potentials were obtained by analysing the solutions in a Malvern Zetasizer 2000 The results are shown in Figure 2.

Example 3 Effect of surface active aqents The bacterial strains used in this study were Escherichia coil W 3110, Bacillus cereus, Enterococcus faecalis and Pseudomonas aeruginosa Cultures were grown in nutrient broth at 37 C with shaking until the optical density at 600 nm was 0 3.

An aliquot of each culture ( 1001 l) was added to 10 ml of 5 m M MES buffer, p H 6 0, containing 5 % sucrose and either 0, 0 005, 0 05 or 0 5 m M CTAB The buffer solution was filtered through a 0 2 pm filter prior to use to remove small particles that may interfere with subsequent electrophoretic measurements.

Electrophoretic mobilities and the derived zeta potentials were obtained by analysing the solutions in a Malvern Zetasizer 2000 The results are shown in Figure 3.

Example 4 Effect of Stains The bacterial strains used in this study were Escherichia coli W 3110, Bacilllus cereus ATCC 11778, Enterococcus faecalis NCTC 12697, Pseudomonas aeruginosa NCTC 6750, Staphylococcus saprophyticus NCTC 7292, Proteus mirabilis NCTC 10374 Cultures were grown in nutrient broth at 37 C, with shaking, until the optical density at 600 nm was 0 3.

An aliquot of each culture ( 100 p I) was added to 10 ml volumes of buffer solution containing 5 m M MES p H 6 0, 5 % sucrose and varying concentrations of either Crystal Violet, Carbol Fuchsin, Methylene Blue, Malachite Green or Safranin.

The buffer solution had been filtered through a 0 2 pm filter prior to use to remove small particles that may interfere with subsequent electrophoretic measurements.

Electrophoretic mobilities and the derived zeta potentials were obtained by analysing the solutions using a Malvern Zetasizer 2000 The results are shown in Figures 4-8.

Example 5 Effect of Chelatinq agents The bacterial strains used in this study were Escherichia coli W 3110, Bacillus cereus ATCC 11778, Enterococcus faecalis NCTC 12697, Pseudomonas aeruginosa NCTC 6750, Staphylococcus saprophyticus NCTC 7292, Proteus mirabilis NCTC 10374 Cultures were grown in nutrient broth at 37 C, with shaking, until the optical density at 600 nm was 0 3.

An aliquot of each culture ( 100 11) was added to 10 ml of buffer solution containing 5 m M MES p H 6 0, 5 % sucrose and either 0, 0 1, 1 0, or 5 m M EDTA.

The buffer solution had been filtered through a 0 2 pm filter prior to use to remove small particles that may interfere with subsequent electrophoretic measurements.

Electrophoretic mobilities and the derived zeta potentials were obtained by analysing the solutions using a Malvern Zetasizer 2000 The results are shown in Figure 9.

Example 6 Effect of p H chanae The bacterial strains used in this study were Escherichia coli W 3110, Bacillus cereus ATCC 11778, Enterococcus faecalis NCTC 12697, Pseudomonas aeruginosa NCTC 6750, Staphylococcus saprophyticus NCTC 7292, and Proteus mirabilis NCTC 10374 Cultures were grown in nutrient broth at 37 C, with shaking, until the optical density at 600 nm was 0 3.

An aliquot of each culture ( 100 11) was added to 10 ml volumes of 2 5 m M citrate buffer at p H 3, 4, 5, 6 and 6 5 The conductivity of the buffer solutions was maintained at 0 7 m S by the addition of Na CI The buffer solutions had been filtered through a 0 2 Lm filter prior to use to remove small particles that might interfere with subsequent electrophoretic measurements Electrophoretic mobilities and the derived zeta potentials were obtained by analysing the solutions using a Malvern Zetasizer 2000 The results are shown in Figure 10.

Example 7 Effect of Conductivity The bacterial strains used in this study were Escherichia coil W 3110, Bacillus cereus ATCC 11778, Enterococcus faecalis NCTC 12697, Pseudomonas aeruginosa NCTC 6750, Staphylococcus saprophyticus NCTC 7292, Klebsiella aerogenes NCTC 10004, and Proteus mirabilis NCTC 10374 Cultures were grown in nutrient broth at 37 C, with shaking, until the optical density at 600 nm was 0 3.

An aliquot of each culture ( 100 pl) was added to 10 ml volumes of 5 m M sodium phosphate buffer p H 7 0 containing either 0, 1 5, 3, 4 5, or 6 m M Na CI The buffer solution had been filtered through a 0 2 lim filter prior to use to remove small particles that might interfere with subsequent electrophoretic measurements.

Electrophoretic mobilities and the derived zeta potentials were obtained by analysing the solutions using a Malvern Zetasizer 2000 The results are shown in Figure 11.

Example 8 Effect of different buffers The bacterial strains used in this study were Escherichia coli W 3110, Bacillus cereus ATCC 11778, Enterococcus faecalis NCTC 12697, Pseudomonas aeruginosa NCTC 6750, Staphylococcus saprophyticus NCTC 7292 and Proteus mirabilis NCTC 10374 Cultures were grown in nutrient broth at 37 C, with shaking, until the optical density at 600 nm was 0 3.

An aliquot of each culture ( 1001) was added to 10 ml volumes of 5 m M citrate buffer p H 3 0, 5 m M acetate buffer p H 4 0, 5 m M MES buffer p H 6 0, 5 m M sodium phosphate buffer p H 7 0 and 5 m M Tris-HCI buffer p H 8 0 The buffer solutions had been filtered through a 0 2,pm filter prior to use to remove small particles that might interfere with subsequent electrophoretic measurements.

Electrophoretic mobilities and the derived zeta potentials were obtained by analysing the solutions using a Malvern Zetasizer 2000 The results are shown in Figure 12.

Example 9 Effect of two polvols: Polyethylene qlvcol (PEG) 4000 and Sorbitol The bacterial strains used in this study were Escherichia coli W 3110, Bacillus cereus ATCC 11778, Enterococcus faecalis NCTC 12697, Pseudomonas aeruginosa NCTC 6750, Staphylococcus saprophyticus NCTC 7292, Klebsiella aerogenes NCTC 10004 and Proteus mirabilis NCTC 10374 Cultures were grown in nutrient broth at 37 C, with shaking, until the optical density at 600 nm was 0 3.

An aliquot of each culture ( 100 pl) was added to 10 ml of buffer solution containing 5 m M MES p H 6 0 and either 0, 2 5, 5 0, 7 5, or 10 0 % of PEG 4000 or sorbitol The buffer solutions had been filtered through a O 2 p Lm filter prior to use to remove small particles that may interfere with subsequent electrophoretic measurements Electrophoretic mobilities and the derived zeta potentials were obtained by analysing the solutions using a Malvern Zetasizer 2000 The results are shown in Figures 13 and 14.

Example 10 Effect of Heavy metal salts The bacterial stains used in this study were Escherichia coli W 3110, Bacillus cereus ATCC 11778, Enterococcus faecalis NCTC 12697, Pseudomonas aeruginosa NCTC 6750, Staphylococcus saprophyticus NCTC 7292, Proteus mirabilis NCTC 10374 Cultures were grown in nutrient broth at 37 C, with shaking, until the optical density at 600 nm was 0 3.

An aliquot of each culture ( 100 Ol) was added to 10 ml of buffer solution containing 5 m M MES p H 5 0, 5 % sucrose and 100 m M of either Nickel chloride, Copper chloride or Lead nitrate The buffer solutions had been filtered through a 0.2 rpm filter prior to use to remove small particles that may interfere with subsequent electrophoretic measurements Electrophoretic mobilities and the derived zeta potentials were obtained by analysing the solutions using a Malvern Zetasizer 2000.

The results are shown in Table 2.

Table 2 Effect of heavy metal salts on bacterial zeta potential Zetapotential m V No heavy Ni C 12 6 H 20 Cu CI 2 2 H 20 Pb(N 03)2 metal E coli -45 2 -31 8 -30 7 -24 0 E faecalis -39 0 -32 9 -35 1 -32 5 Ps aeruqinosa -33 1 -27 9 -28 9 -27 0 Staph -39 1 -30 7 -31 0 saprophyticu S P mirabilis -38 2 -32 7 -31 6 -27 0 Example 11 Detection of virus byzetapotential measurements The virus used in this study was the bacteriophage T 4 T 4 was prepared by inoculating an E coli W 3110 culture, grown in phage broth at 37 C to an optical density of 600 of 0 3, with Iml of phage lysate After approximately 2 hours incubation at 370 C, 'clearing' of the culture occurred and 5 ml of chloroform was added The culture was shaken, allowed to settle and the chloroform layer decanted The remaining phage lysate was further purified by standard procedures as described by Sambrook, J, E F Fritsch, and T Maniatis, ( 1989) Molecular cloning: a Laboratory Manual 2nd Edition Cold Spring Harbor Press, New York.

An aliquot ( 100 p I) of purified T 4 phage suspension was added to 10 ml of filtered buffer containing 5 m M MES p H 6 0 and 5 % sucrose Electrophoretic mobility and the derived zeta potential were obtained by analysing the solution using a Malvern Zetasizer 2000 The results are shown in Table 3.

Table 3 Zetapotential of Bacteriophaqe T 4 Virus Zetapotential (m V) Bacteriophage T 4 -34 6 Example 12 Effect of Lvsozvme The bacterial stains used in this study were Escherichia coli W 3110, Bacillus cereus ATCC 11778, Enterococcus faecalis NCTC 12697, Pseudomonas aeruginosa NCTC 6750, Staphylococcus saprophyticus NCTC 7292, Proteus mirabilis NCTC 10374 Cultures were grown in nutrient broth at 37 C, with shaking, until the optical density at 600 nm was 0 3.

An aliquot of each culture ( 100 pl) was added to 10 m L of buffer solution containing 5 m M MES, p H 6 0, 5 % sucrose and either 0, 5, 10, 15, 20 gig/ml of mutanolysin Reactions were incubated at 37 C for 30 min The buffer solution had been filtered through 1 0 2 pim filter prior to use to remove any small particles that may interfere with subsequent electrophoretic measurements.

Electrophoretic mobilities and derived zeta potentials were obtained by analysing the solutions using a Malvern Zetasizer 2000 The results are shown in Figure 15.

Example 13 Effect of Mutanolvsin The bacterial stains used in this study were Escherichia coli W 3110, Bacillus cereus ATCC 11778, Enterococcus faecalis NCTC 12697, Pseudomonas aeruginosa NCTC 6750, Staphylococcus saprophyticus NCTC 7292, Proteus mirabilis NCTC 10374 Cultures were grown in nutrient broth at 37 C, with shaking, until the optical density at 600 nm was 0 3.

An aliquot of each culture ( 100 i 1) was added to 10 m L of buffer solution containing 5 m M MES, p H 6 0, 5 % sucrose and either 0, 10, 25 or 50 U/ml of mutanolysin Reactions were incubated at 37 C for 30 min The buffer solution had been filtered through 1 0 2 lm filter prior to use to remove any small particles that may interfere with subsequent electrophoretic measurements Electrophoretic mobilities and derived zeta potentials were obtained by analysing the solutions using a Malvern Zetasizer 2000 The results are shown in Figure 16.

Only E coli shows a significant change in the presence of mutanolysin.

Example 14 Effect of the Antibacterial Peptide Cecropin The bacterial stains used in this study were Escherichia coil W 3110, Bacillus cereus ATCC 11778, Enterococcus faecalis NCTC 12697, Pseudomonas aeruginosa NCTC 6750, Staphylococcus saprophyticus NCTC 7292, Proteus mirabilis NCTC 10374 Cultures were grown in nutrient broth at 37 C, with shaking, until the optical density at 600 nm was 0 3.

An aliquot of each culture ( 100 pl) was added to 10 m L of 1 m M sodium phosphate buffer, p H 7 0, containing either 0 or 5 g/ml cecropin The buffer solution had been filtered through 1 0 2 gm filter prior to use to remove any small particles that may interfere with subsequent electrophoretic measurements.

Electrophoretic mobilities and derived zeta potentials were obtained by analysing the solutions using a Malvern Zetasizer 2000 The results are shown in Table 4.

Table 4 Effect of cecropin on bacterial zeta potential.

Bacterial strains Zeta potential Zeta potential + Cecropin Cecropin Change (%) E coli -51 8 -42 8 21 0 B cereus -33 9 -33 5 1 2 Ent faecalis -28 1 -27 5 2 2 Ps aeruginosa -56 5 -20 1 181 Staph aureus -43 9 -44 1 1 P mirabilis -42 1 -41 9 1 E coil and Ps Aeruginosa both show a large change in zeta potential; the other bacteria show no change.

Claims (14)

Claims

1 A method of identifying one or more micro-organisms in a fluid sample, said method comprising the steps of:- (i) optionally culturing the sample if necessary to increase the number of micro- organisms to a pre-determined range; (ii) applying an electric field across a portion of said fluid; (iii) measuring the velocity, displacement, zeta potential or electrophoretic mobility of any micro-organisms present following the application of said electric field; (iv) re-measuring the velocity, displacement, zeta potential or electrophoretic mobility of the fluid sample after incubation in the presence of a bioactive peptide; (v) comparing said measured velocities, displacements, zeta potentials or electrophoretic mobilities with tables of velocities, displacements, zeta potentials or electrophoretic mobilities of known micro-organisms measured under substantially identical experimental conditions to determine which, if any, micro-organisms are present.

2 A method according to Claim 1 wherein the bioactive peptide is selected from the group consisting of lysozyme, mutanolysin, lyticase, chitinase, mucopeptide amidohydrolase, N-acetylglucosamine deacetylase, B( 1-6) and 8 ( 1-3) glycanases, proteases, mannase, cecropins and cecropin-like proteins, Magainin I and II, amide, Cecropin P 1, A and B, Defensin HNP-1 and HNP-2, Nisin, Lactoferricin, Buforin II, indolocidin, ranalexin, sarcotoxins, lepidopterans, moricins and hyphancins.

3 A method according to Claim 1 or Claim 2 wherein the bioactive peptide is lysozyme.

4 A method according to Claim 1 or Claim 2 wherein the bioactive peptide is mutanolysin.

A method according to Claim 1 or Claim 2 wherein the bioactive peptide is cecropin or a cecropin-like peptide.

6 A method according to Claim 1 or Claim 2 wherein the bioactive peptide is magainin.

7 A method according to Claim 1 or Claim 2 wherein aliquots of the fluid sample produced at the end of optional stage (i) are incubated with or suspended in an array of different bioactive peptides, including at least one control without bioactive peptide present.

8 A method according to any preceding claim wherein in optional step (i) the sample is cultured in a medium selective for a particular organism or class of organisms, said modification providing a method of distinguishing between micro- organisms that otherwise have similar velocities, displacements, zeta potentials or electrophoretic mobilities.

9 A method according to any preceding claim wherein the comparison between the measured velocity displacement, zeta potential or electrophoretic mobility and said table of known velocities, displacements, zeta potentials or electrophoretic mobilities is carried out using a computer program, the result being displayed on a computer screen or by way of a printout.

A method according to any preceding claim wherein said fluid sample is obtained from a human or animal body and said method provides an indication of the cause of an infection in said human or animal body.

11 A method according to any preceding claim wherein aliquots of the fluid sample containing the micro-organism(s) are, in addition to incubation with one or more bioactive peptide, suspended in a plurality of different solutions, said solutions representing different experimental conditions differing in one or more of the parameters selected from the group of parameters consisting of p H, ionic strength, concentration of surface-active agent, concentration of heavy metal ions, concentration of chelating or complexing agent, concentration of organic solvent, concentration of dye or stain, concentration of polyol, or concentration of polyether; and subsequently comparing said measured velocities, displacements, zeta potentials or electrophoretic mobilities with tables of velocities, displacements, zeta potentials or electrophoretic mobilities of known micro-organisms measured under substantially identical experimental conditions to determine which, if any, micro- organisms are present.

12 A method of identifying one or more micro-organisms in a fluid sample substantially as herein described.

13 An apparatus for carrying out the methods described in any of Claims 1 to 12 inclusive comprising:- (i) means for applying an electric field across a measurement cell; (ii) a light source for illuminating the measurement cell; (iii) detecting means for detecting light scattered by micro-organisms present in the cell; (iv) means for analysing the scattered light to provide a measurement of the speed of movement of a micro-organism(s); (v) means for computing the velocity, displacement, zeta potential or electrophoretic mobility of said micro-organism(s); (vi) means for comparing said measured velocities, displacements zeta potentials or electrophoretic mobilities, with the velocities, displacements, zeta potentials or electrophoretic mobilities of known organisms; characterised in that the apparatus further comprises an array of measurement cells containing one or more bioactive peptides selected from the group comprising lysozyme, mutanolysin, lyticase, chitinase, mucopeptide amidohydrolase, N-

acetylglucosamine deacetylase,,( 1-6) and,?( 1-3) glycanases, proteases, mannase, cecropins and cecropin-like proteins, Magainin I and II, amide, Cecropin P 1, A and B, Defensin HNP-1 and HNP-2, Nisin, Lactoferricin, Buforin II, indolocidin, ranalexin, sarcotoxins, lepidopterans, moricins and hyphancins.

14 An apparatus according to Claim 13 wherein the array of measurement cells contains, in addition to one or more bioactive proteins, a plurality of different solutions, said solutions representing different experimental conditions differing in one or more of the parameters selected from the group of parameters consisting of p H, ionic strength, concentration of surface-active agent, concentration of heavy metal ions, concentration of chelating or complexing agent, concentration of organic solvent, concentration of dye or stain, concentration of polyol, or concentration of polyethers.

An apparatus for carrying out the methods described in Claims 1 to 12 inclusive substantially as herein described.