WO2004111264A1

WO2004111264A1 - Bacterial biofilm assay employing magnetic beads

Info

Publication number: WO2004111264A1
Application number: PCT/CA2004/000887
Authority: WO
Inventors: Michael G. Surette
Original assignee: University Technologies International Inc.
Priority date: 2003-06-13
Filing date: 2004-06-14
Publication date: 2004-12-23

Abstract

A biofilm assay method includes the steps of growing a micro organism in a culture medium having at least one magnetic bead, such that the micro organism grows as a biofilm on the magnetic bead, and using a magnet to manipulate the magnetic bead.

Description

BACTERIAL BIOFILM ASSAY EMPLOYING MAGNETIC BEADS

Background of the Invention

Bacteria in nature commonly grow attached to solid surfaces in what is referred to as a biofilm. Biofilms may exhibit much greater resistance to antibiotics than those grown suspended in liquid culture (planktonic cells). Persistent and chronic infections refractory to treatment with conventional antibiotics are involved with 60% of bacterial infections, according to estimates by the U.S. Centers for Disease Control. Industrially, biofilms contaminate and clog water lines, foul surfaces and contribute to corrosion and decay. However, not all the consequences of biofilm formation are deleterious. For example, in bioproduction processes, biofilms help in maintaining a stable population of cells as substrate passes through a bioreactor.

These clinical and industrial ramifications necessitate a search for compounds that influence biofilm formation. Current methods of finding such compounds entails screening libraries of thousands of candidates to maximize the chance of finding a useful one. Such high-throughput screening, in turn, generates a need for reliable, convenient methods for analyzing large numbers of samples quickly and cheaply. Furthermore, because biofilms form on a variety of surfaces, the high-throughput screening methodology ideally would permit facile modification of the surfaces on which the biofilm-forming bacteria grow.

Previously, others have attempted to study biofilms through a variety of methods. Flow cells, the traditional approach for growing and characterizing biofilms, involves flowing medium over a surface on which the biofilm will grow. Typically the flow cell includes an optically clear portion to allow examination of the growing biofilm by microscopy, after removal of planktonic cells by washing. For example, U.S. Patent No. 5,641,458 to Shockley, Jr. et al. discloses a flow cell for non-invasively monitoring bodily fluids that includes sensors that interact with a fluid sample through a semi-permeable membrane, and allow the fluid sample to be monitored optically. Such flow cells do not permit many samples to be investigated at a time, and hence are not suitable for high-throughput screening. U.S. Patent No. 5,624,815 to Grant et al discloses a method and apparatus for analyzing biological material by drawing a liquid sample through a number of discrete wells that retain the biological material. Also, U.S. Patent No. 5,792,430 to Hamper, U.S. Patent No. 5,624,815 to Grant et al, U.S. Patent No. 4,908,319 to Smyczek et al, and U.S. Patent No. 4,753,775 to Ebersole et al all disclose ways of studying biological material in which a liquid sample is drawn over a solid support.

Another approach involves growing biofilms on polystyrene plates. However, this methods does not allow for continuous monitoring of the biofilm because the biofilm is characterized by staining at a single time and is thereby destroyed. The limited flow of medium over the polystyrene surface may also yield biofilms with different properties than those grown in flow cells. Nevertheless, this method provides higher assay throughput than that possible with use of flow cells.

Still another device attempts to increase throughput screening of biofilms through use of a plate lid with pins that insert into medium contained in each well of the plate. In one form, the plates have troughs that interconnect the wells. Rocking the plate then causes medium to flow over the surfaces, and is thought to produce a biofilm more like that observed in flow cells. These devices are expensive and provide little scope for modifying the surfaces on which the bacteria grow. Furthermore, quantitating the resulting biofilms necessitates their disruption, so that biofilm development cannot be followed over time.

More recently biofilms have been grown on glass beads in trays, where again gentle shaking of the trays provides fluid flow like that for the pin device. While this approach could be used with polystyrene beads that permit modifications to the surface, neither glass nor polystyrene beads readily lend themselves to the manipulations involved in high-throughput screening because manipulating the beads to move them typically damages the biofilms on them.

There is thus a need for convenient methods of assaying the formation and maintenance of biofilms in a relatively inexpensive manner to facilitate development of agents for controlling them. Such methods may allow for high-throughput assays. The methods may preferably be amenable to automation, and in particular to allowing manipulation of the biofϊlm-laden surface without damaging the biofilm. Further, the methods may preferably permit study of the time course of biofilm formation and/or maintenance without damaging the biofilm. It is also desirable that the methods lend itself to modification of the surface on which the biofilm forms.

Summary of the Invention

In one aspect, the invention may comprise a method of manipulating biofilms, comprising the steps of growing a microorganism in a first culture medium having at least one magnetic bead, such that the microorganism grows as a biofilm on the magnetic bead, and using a magnet to move the magnetic bead relative to the first medium.

The biofilm on the magnetic bead may then be assayed in any manner, hi one embodiment, the assaying method may be an optical method or a plating method. Suitable optical methods may include light scattering, fluorescence spectroscopy, luminescence spectroscopy, and absorption spectroscopy. A suitable luminescence spectroscopic method may include measuring the bioluminescence of a lux-based reporter. A suitable absorption spectroscopic method may include a colorimetric determination of a reporter fused to a biofilm-specific or a constitutive promoter.

In one embodiment, the magnet is used to separate the bead from the first medium. The bead is returned to the first medium or transferred to a second culture medium.

The beads comprise a magnetic material and may have a diameter of about 10 μm to 500 μm. In a preferred embodiment, the bead may have a diameter of about 100 to 500 μm which may more preferably be a diameter of about 100 to 200 μm.

The beads may be coated or uncoated. If coated, the bead may comprises a surface material such as polystyrene and polycarbonate. The surface of the bead may be chemically-modified such as by treatment with a crosslinking reagent such as a reagent comprising a carbodiimide functional group. In another aspect, the invention may comprise an assay kit for assaying a biofϊlm property, said kit comprising:

(a) at least one multiwell growth plate;

(b) at least one magnetic bead;

(c) a growth media; (d) means for assaying properties of a biofϊlm comprising a microorganism on the at least one magnetic bead.

In another aspect, the invention may comprise an improvement to a high-throughput assay for the formation or maintenance of a biofϊlm, comprising the steps of growing a microorganism in a first culture medium having at least one magnetic bead, such that the microorganism grows as a biofϊlm on the magnetic bead, and using a magnet to move the magnetic bead relative to the first medium.

Brief Description of the Figures

The present invention will now be described with reference to the following figures.

Figure 1 shows one example of a standard wash procedure for screening.

Figure 2 shows a schematic procedure for evaluating washing efficiency.

Figure 3 shows biofϊlm and cell aggregates present in a microtitre assay. The schematic on the left represents a freshly inoculated microtitre well (bacteria represented as red cells) with magnetic beads added (4 large beads). The schematic on the right represents the culture after a period of growth (e.g. 24 h) Each group of cells likely represents unique states with the surface-attached cells representing the most heterogeneous mixture of cellular states.

Figure 4 shows adhered cells to magnetic bead biofilm transfer device after successive washes. Pellicle cells may adhere to the surface of the transfer device depending on the strain and growth conditions. Figure 5 shows the reporter vector pMS402 containing a promoter-less luxCDABE reporter and a low copy origin of replication for Pseudomonas aeruginosa, as an example of a reporter system for real-time gene expression analysis.

Figure 6 shows graphs demonstrating washing efficiency and biofilm formation on magnetic bead biofilms. Pseudomonas aeruginosa strain PAOl containing a reporter plasmid for the oprH promoter (driving luxCDABE expression in pMS402).

Figure 7 shows values for the triplicate assays shown in Figure 6 for the last wash (Wash 7) and magnetic bead biofilms. No normalization for bead number was made.

Figure 8 shows light microscopy of magnetic bead biofilms of Pseudomonas aeruginosa mono cultures at 24 h.

Figure 9 shows light microscopy of sputum samples comparing mixed species aggregates differing in appearance from flow cell grown biofilms and more closely resembling magnetic bead biofilm.

Description of Embodiments of the Invention

The present invention provides a biofilm assay method and systems that use magnetic beads as the surface on which biofilm formation takes place. The term "magnetic bead" designates a bead or particle that is attracted to, or repelled by, a magnetic field. A magnetic bead may or may not be magnetized before exposure to the magnetic field. Reference to the term "magnafilm" herein shall be a reference to a biofilm or bioaggregate growing on a magnetic bead.

As used herein, a "biofilm" forms when microorganisms adhere to surfaces in an aqueous environment and grow to cover a substantial portion of the surface. A biofilm may begin to excrete substances that can anchor them to a substrate material - such as metals, plastics, soil particles, medical implant materials, and tissue. A biofilm can be formed by a single bacterial species, or may comprise of many species of bacteria, as well as fungi, algae, protozoa, debris and corrosion products. A biofilm may form on any surface exposed to bacteria and some amount of water.

In general terms, the practice of the present invention involves the use of one or more magnetic beads which are placed in a receptacle containing a culture medium in which the bacteria of interest can grow. Then, the bead or beads may be manipulated or separated from the medium through the use of a magnetic field. Subsequently or concurrently, the extent of any biofilm that may have formed, or any other property of the biofilm, may then be assayed.

hi one embodiment, the beads may be removed from the medium by introducing a magnet into the medium until the bead adheres to the magnet, at which point the user can withdraw the magnet with the bead attached. In one embodiment, such bead transfers may involve the use of arrays of pin magnets, so that a magnet will dip into each well of a plate having a plurality of wells. The magnetic pin arrays may be configured as 96 or 384 pin arrays in order to match standard commercially available plates.

In an alternative embodiment, the beads can be separated from the culture medium by placing a magnet adjacent to the receptacle such that the magnetic bead adheres to a wall or the bottom of the receptacle. The culture medium may then be removed, for example, by inverting the receptacle or aspirating the medium from the receptacle. The aspiration of liquid media from receptacles maybe automated using known systems.

Once the beads have been separated from the culture medium the user can then measure any property of any biofilm that may have formed on the bead, for example, the extent of biofilm formation, or gene expression levels, through methods well-known in the art (Valdivia, R. H. and Falkow, S., 1997; Van Dyk, T. K.; DeRose, E. J.;Gonye, G. E., 2001; Goh, E. B.; Yim, G.; Tsui, W.; McClure, J.; Surette, M. G.;Davies, J., 2002; Kalir et al., 2001).

The user can also make determinations on the free-living bacteria in the culture, or in the culture medium itself, in either case free of the bead and the bacteria living in the biofilm. The ability to make such determinations repeatedly allows the user to follow a property as a function of time, so that, for example, the user can determine gene expression profiles for both the bacteria in the biofilm and the planktonic bacteria (free-living bacteria suspended in the culture medium).

Properties that can be measured through standard methods include determination of differentially regulated genes, for example, through use of promoter libraries, quantitation of cells indirectly by protein/DNA staining. For assay development, cells can also be quantitated, for example, directly by commonly-used plating methods or 16 S PCR/DGGE or selective plating, evaluation of antagonists and high-throughput screening of potential inhibitors and chemical libraries, and measurement of cell numbers through fluorescent staining of DNA or lipids. This enumeration of measurable properties is not intended to be limiting of the claimed invention.

Mixed bacterial communities may be characterized by fluorescent Gram staining (agents for which are available from Molecular Probes, Eugene, OR) and microscopy. Bioaggregates on the beads may be characterized by scanning electron microscopy or by confocal microscopy of beads differentially stained with, for example, fluorescent Gram stain. Similarly, in other examples, direct plating on selective media can be used for cell enumeration and lux reporter assays can be used to measure effects on gene expression on one strain that arise from interaction with other species in a mixed community.

Bead size balances available surface area with easy manipulation by magnets. The surface area of the beads determines the area of biofilm which may form. In one embodiment, bead diameters of about 100-500 μm may be suitable. Beads of 100 μm diameter have surface area of approximately 50,000 μm² per bead; while those of 500 μm diameter have surface area of about 1 mm per bead, which if covered in a monolayer of cells would equal approximately 500,000 cells, equivalent to approximately 5 μL of a bacterial culture in mid log phase of growth. Such a biofilm is readily detectable with a variety of detection systems to measure bacteria or bacterial gene expression. A monolayer represents the theoretical lower limit of cell numbers because biofilms may grow in three dimensions from the surface of attachment and do not necessarily exist as a monolayer of cells.

One embodiment of a biofilm assay of the present invention preferably uses beads of about 100 μm diameter. For example, magnetic beads having a size distribution between about 75 to about 106 μm are available from Spherotech Inc., Libertyville, IL. Measurements on bacterial cell associations not attached to a substrate (also known as bioaggregate) preferably use magnetic beads of intermediate size of about 50 μm,such as the commercially available 32-53 μm and 53-75 μm beads. Such beads are large enough for biofilm formation and yet small enough for manipulation with magnets. While larger beads can be used, magnetic manipulation of beads becomes less convenient as their size increases.

Magnetic beads useful in practicing the present invention include without limitation those beads that exhibit ferromagnetic, ferrimagnetic, or paramagnetic behavior, hi one embodiment ferromagnetic beads maybe used, such as those composed of hematite or of a superparamagnetic alloy, such as cobalt rare earth alloys. Methods of magnetizing magnetic materials, such as ferromagnetic ones, are well known in the art. The beads may be uncoated or coated with a suitable material such as polystyrene, rubber latex, polycarbonate, polyvinyl alcohol, dextran, silica, carbon, or other suitable materials. Multiple coating layers or composite coating layers may be utilized for specific purposes.

Many bacteria adhere to surfaces through specific ligand-receptor interaction. For example, bacteria that colonize animals bind to extracellular matrix material, such as substances found in the mucous layer of epithelial surfaces. These substances may include mucins, actins, collagens or fibronectins. Many of these substances, particularly mucin, interact with polystyrene and other materials used to make beads. Biofilm assays preferably employ surfaces that promote specific adhesion and biofilm growth, and therefore may require particular surfaces. Consequently, in one embodiment the present invention may provide magnetic beads having chemically-functionalized surfaces such as mucin bound to the bead surface. Magnetic beads are commercially available with a variety of surfaces that lend themselves to chemical derivatization, such as surfaces amenable for coating with extracellular matrix constituents. For example, in one embodiment, beads bearing surface carboxyl groups may be treated with chemical crosslinking reagents to generate surface-modified beads bearing stable, defined surfaces. Suitable crosslinking agents may include, without limitation, carboiimides and carbodiimide-sulfydryl specific groups. Other crosslinking agents are well-known in the art. In another embodiment, the beads can have pendant amino or sulfhydryl groups. Addition of mucin, fibronectin, collagen, or other extracellular matrix material to surface-modified beads bearing ammonium or carboxyl groups, followed by a crosslinking reagent, and then washing with sterile buffer, affords surface modified beads suitable for use in assays. One example of a crosslinking reaction may involve incubation with dicyclohexylcarboiimide for 10-15 minutes at room temperature,

Magnetic beads may be coated without use of chemical crosslinking reagents to keep the structural integrity of the matrix intact. For example, mucin may interact with a bead surface through electrostatic and hydrophobic interactions strongly enough to persist through most assays. Chemically crosslinking pendant groups on the bead surface can also modify any extracellular matrix materials present there, which can adversely affect its ability to interact with the microbe. While in practice cells often adhere to beads with charged surfaces {e.g., those bearing protonated amino groups) without the need for extracellular matrix, coated beads may be treated with crosslinking reagents to increase the stability of the coating, if desired.

Assays suitable for use with the present invention include those based on optical methods and plating methods. Optical methods include those assay methods which involve the detection or measurement of electromagnetic radiation in the visible spectrum and may include luminescence and absorption spectroscopy. Plating methods include those assay methods which may measure bacterial numbers using selective or non-selective media.

hi one embodiment, an optical method such as a luminescence spectroscopic method may employed. Such methods can be advantageously employed through incorporation into the bacterial strain of interest of a reporter vector such as pMS402 containing a promoter-less reporter, such as luxCDABE The use of such a reporter is described in US Patent No. 6,559,176, the contents of which are incorporated by reference in its entirety. hi one embodiment using a lux reporter, the bacterial strain may be grown in the presence of magnetic beads, which may or may not be surface modified, under desired conditions. The magnetic beads are then transferred to fresh medium in 96 well plates (as described below) and luminescence measured with standard well-known instruments, such as a Wallac Model 1409 liquid scintillation counter (Wallac hie, Gaitliersburg, MD). hi this case, light production correlates to the number of cells for constitutively expressed reporters.

hi another embodiment, a colorimetric method of absorption spectroscopy may be employed. One way of carrying out such a colorimetric method is to insert into the desired bacterial strain a reporter vector containing a reporter that generates a measurable adsorbent when it acts upon a substrate. Such reporters are well-known to those skilled in the art and may include beta-galactosidase. The magnetic beads may then be transferred to fresh medium in 96 well plates (as described below). Cells are lysed and enzymatic activity measured through use of standard procedures. For example, in an assay for beta- galactosidase, cells are lysed with 0.1% SDS and chloroform and enzyme levels quantitated through use of a colorigenic substrate such as 2-nitrophenyl-β-D- galactopyranoside. The absorbance measured correlates to enzyme levels (for constitutively expressed reporters) that in turn correlate to the number of cells.

Plating methods for quantitating biofilm growth depend upon measuring cell densities through diluting aliquots of cells, spreading the dilutions onto solid medium , incubating the plates , and later counting the resulting colonies. Magnetic beads are transferred to fresh medium and sonicated, or otherwise treated, to release cells from the biofilm/magnetic bead. The cell suspension is then diluted into sterile medium, and aliquots are plated onto selective solid medium for enumeration of the colonies that result. Suitable selective media are well-known in the art and may include Pseudomonas isolation agar (Difco Cat# 0927-17) for P. aeruginosa and Columbia CNA Agar (Difco Cat # 212104) for Gram-positive bacteria.

The assay methods of the present invention may be adapted to include the use of robots, thereby facilitating high-throughput screening by minimizing the tedious task of performing bead manipulations manually. While permanent magnets can be used to manipulate the beads, it is also possible to use electromagnets which may facilitate such automated methods.

Examples

The following examples are intended to illustrative of the claimed invention, but not limiting of the claimed invention.

Standard procedures for magnetic bead manipulations on liquid-handling robots {e.g., on the Beckman CoulterBiomek® 2000 or the Packard MultiProbe-HTS®) equipped with magnetic separators (such as the Dynal MPC-auto-96, a magnetic particle concentrator on a 96-well format that can be switched on and off by the robot software) may be adapted to the study of biofilms with use of magnetic beads.

Magnetic bead manipulations may be performed through use of a Packard Bioscience Multiprobe II™ or Beckman BioMek2000™ outfitted with a 384 well pipetting head, magnetized platform, and gripper tool all designed specifically for use with these liquid- handling robots.

Previously arrayed P. aeruginosa independent clones containing the random promoter library were inoculated to 384 well conical test plates for development of bio-aggregates or biofilms. Streptococcus and Staphylococcus spp. or other appropriate bacteria were dispensed to a P. aeruginosa library containing wells for mixed-species work. Magnetic beads treated with matrix were then dispensed, by pipetting, to mono- or mixed-species planktonic bacterial cultures in test plates. Cultures were then incubated to allow formation of bioaggregates or biofilms on the beads. Following incubation, test plates were transferred to a magnetic platform through use of the gripper arm and the planktonic cultures were separated from magnetic beads by aspiration. Immobilized bio-aggregate coated magnetic beads were washed three times with growth medium. Magnetic bead containing plates were then moved off of the magnetic platform and beads were removed by pipetting to a fresh plate for additional washing, if desired. Removal of the beads from the culture plate to a fresh plate allows separation of beads containing bio-aggregate from biofilms that may have formed directly on culture plates. If desired, bead-containing plates are moved back to the magnetic platform through use of the robotic gripper for additional washing. At this point the analysis of bio-aggregates that have formed on matrix coated magnetic beads can proceed.

Magnetic bead biofilm assays: The following outlines the details of the magnetic bead biofilm assay along with data illustrating its specificity and utility.

Magnetic beads (150-180 μm, amino-magnetic particles XL, available from Spherotech Cat# AMX1600), which are supplied as a dry powder, were suspended in medium or buffer as desired for crosslinking.

Crosslinking Treatment

While Pseudomonas aeruginosa forms biofilms on the amino-activated surface without crosslinking, other bacteria such as Viridans Group Streptococcus and Staphylococcus aureus adhere better to mucin-treated beads. Mucin also increases the viability of the Gram-positive bacteria in mixed biofilms with Pseudomonas.

Crosslinking Reactions:

• 0.9 ml cross-linking buffer (0.05 M MES, pH 6)

• ~50 mg amino-magnetic particles XL, Cat# AMXl 600 o 0.1 ml mucin (5% in ddH₂ O)(Sigma M1778, Type III porcine)

• 100 μl of a fresh solution of 10 mg/ml l-ethyl-3-(3- dimethylaminopropyl)carbodiimide, • hydrochloride in dimethyl sulfoxide • incubate 2 h at room temperature with gentle shaking.

• Wash 3 X with ddH₂ O (1 ml) using magnet to retain beads

• Wash 2 X with 25 mM Tris-HCl pH 7.2

• Resuspend in final volume of 500 μl 10% EtOH

• Store at 4 ⁰C

Alternatively the reaction can be started with the mucin alone for 5 minutes to activate, followed by the addition of the amino-magnetic particles.

Magnetic Bead Biofilm Growth Conditions

In a 96-well sterile microtitre plate add:

• 1-2 μl of amino-magnetic particles • 150 μl of 1/300 dilution of overnight culture of strain(s) in medium + selective antibiotics

• Incubate with shaking at 37 ⁰C

For Pseudomonas aeruginosa, a rich broth such as BHI (Brain Heart Infusion medium [Difco]) or THY (Todd Hewitt Broth [Difco]) supplemented with 0.2% yeast extract [Difco]) may be used. For Viridans Group Streptococcus species and Staphylococcus aureus, THY (Todd Hewitt Broth [Difco] supplemented with 0.2% yeast extract [Difco]) or ¹A THY (diluted with ddH₂ O) may be used.

Washing and Screening Magnetic Bead Biofilms

At selected times the magnetic beads were removed from the assay, washed with growth medium + 0.1 % SDS and transferred to fresh medium. This level of detergent reduces the transfer of non-attached cells and does not affect bacterial viability (most bacteria will grow in up to 1% SDS). At this point the level of cells and promoter activity can be determined as described below. Two wash procedures may be used and are shown schematically in Figures 1 and 2. As seen in Figure 1, magnetic bead biofilms are transferred from microtitre well to 5 successive washes (10 sec each with gentle agitation). Washes 1-4 contain 0.1% in growth medium, Wash 5 is simple growth medium. The magnetic bead biofilms are released to the wells of a black microtitre plate and a property such as luminescence may be measured.

As seen in Figure 2, magnetic bead biofilms are transferred from the growth plate and washed serially in the wells of a microtitre plate for 3 sec with gentle agitation. Wells 1-5 contain growth medium + 0.1% SDS. Wells 6 and 7 contain growth medium. The magnetic biofilm beads are released into wells (sample). Luminescence is measured for all wells.

Background transfer can occur in the absence of beads because of pellicle formation. Washing of beads removes not only planktonic cells but also pellicle cells.

Assay Results

The following results are with Pseudomonas aeruginosa PAOl with an oprH- luxCDABE promoter-reporter fusion as depicted in Figure 5. The oprH gene, which is regulated as part of the PhoPQ regulon, encodes an outer membrane protein involved in cationic peptide resistance.

Note that lux reporters offer about 100-1000 fold greater sensitivity than gfp based reporters and offers a wider range of expression profiling.

Figure 6 shows graphs demonstrating washing efficiency and biofilm formation on magnetic bead biofilms. Pseudomonas aeruginosa strain PAOl containing a reporter plasmid for the oprH promoter (driving luxCDABE expression in pMS402) The samples were grown 24 h in THY (+ trimethoprim 150 g/ml) at 37⁰C and washed as in Figure 2. Luminescence was measured in a TriLux MicroBeta Scintillation Counter (Wallac) 5 minutes after transfer. No significant change in light production was observed after 30 minutes at 37°C. Each bar represents the average of triplicate measurements and the error bar indicates the standard deviation. The values for the triplicate assays shown in Figure 6 for the last wash (Wash 7) and magnetic bead biofilms are shown in Figure 7. No normalization for bead number was made.

Images

The images shown in Figure 8 represent magnetic bead biofilm particles with attached bacteria (Pseudomonas aeruginosa mono cultures at 24 h). The magnetic bead biofilms were grown at 37°C for 24 h and washed as indicated in Figure 2. The micrographs were taken with a Nikon EF400 microscope with 40 X oil immersion objective under phase contrast illumination. Images were captured with a CoHu black and white video camera using a Matrox video capture card. The cells extend over the surface of the bead and the images represent a narrow Z-section. The matrix extends >30 mm from the bead surface and the biofilm cells are trapped within this matrix.

Figure 9 shows photographs from light microscopy of sputum samples showing mixed species aggregates differing in appearance from flow cell grown biofilms and more closely resembling magnetic bead biofilm.

References

The following references are incorporated herein, as if reproduced herein in their entirety.

Goh, E.B., Yim, G., Tsui, W., McClure, J., Surette, M.G., and Davies, J. (2002). Transcriptional modulation of bacterial gene expression by subinhibitory concentrations of antibiotics. Proc. Natl. Acad. Sd. U. S. A 99, 17025-17030.

Valdivia, R.H. and Falkow, S. (1997). Fluorescence-based isolation of bacterial genes expressed within host cells. Science 277, 2007-2011.

Van Dyk, T.K., DeRose, EJ., and Gonye, G.E. (2001). LuxArray, a high-density, genomewide transcription analysis of Escherichia coli using bioluminescent reporter strains. J. Bacteriol. 183, 5496-5505.

Claims

1. A method of manipulating biofilms, comprising the steps of:

(a) growing a microorganism to grow in a first culture medium containing at least one magnetic bead, such that the microorganism grows as a biofϊlm on the magnetic bead, (b) using a magnet to move the at least one magnetic bead relative to the first medium.

2. The method of claim 1 comprising the further step of applying an assaying method to the biofilm on the at least one magnetic bead.

3. The method of claim 2 wherein the assaying method is an optical method or a plating method.

4. The method of claim 3 wherein assaying method is an optical method and is selected from the group consisting of light scattering, fluorescence spectroscopy, luminescence spectroscopy, and absorption spectroscopy.

5. The method of claim 4 wherein the luminescence spectroscopic method includes measuring the bioluminescence of a lux-based reporter.

6. The method of claim 5 wherein the reporter is luxCDABE.

7. The method of claim 4 wherein the absorption spectroscopic method includes a colorimetric determination of a reporter fused to a biofilm-specifϊc or a constitutive promoter.

8. The method of claim 1 wherein the magnet is used to separate the at least one magnetic bead from the first medium.

9. The method of claim 8 wherein the at least one magnetic bead is returned to the first medium or transferred to a second culture medium.

10. The method of claim 1 wherein the at least one magnetic bead has a diameter of about 10 μm to 500 μm.

11. The method of claim 10 wherein the at least one magnetic bead has a diameter of about l00 to 500 μm.

12. The method of claim 11 wherein the at least one magnetic bead has a diameter ofabout l00 to 200 μm.

13. The method of claim 1 wherein the at least one magnetic bead comprises a surface material comprising polystyrene or polycarbonate.

14. The method of claim 1 wherein the surface of the bead is chemically- modified.

15. The method of claim 14 wherein the chemical modification is treatment with a reagent comprising a carbodiimide functional group.

16. An assay kit for assaying a biofilm property, said kit comprising:

(a) at least one multiwell plate;

(b) at least one magnetic bead;

(c) a growth media; and

(d) means for assaying properties of a biofilm comprising a microorganism on the at least one magnetic bead.

17. In a high-throughput assay for the formation or maintenance of a biofϊlm, an improvement comprising the steps of growing a microorganism in a first culture medium having at least one magnetic bead, such that the microorganism grows as a biofilm on the magnetic bead, and using a magnet to move the magnetic bead relative to the first medium.