AU719315B2 - Method for detecting microorganisms - Google Patents

Description

WO 96/31777 PCT/AU96/00192 1 Method for Detecting Microorganisms Technical Field The present invention relates to methods for detecting the presence of microorganisms in a sample using particles bearing a ligand reactive to the microorganisms and fluorescent labelled ligands. The methods are suitable for flow cytometric detection of microorganisms.

Background Art Testing samples for the presence of microorganisms, in particular human pathogens, is an important part of monitoring samples including biological samples, foods, drinks, the environment and water supplies. In order to obtain immediate results, testing often involves the direct analysis of samples for specific microorganisms. This can be labour intensive and routine for the technician involved. In particular, there is an increasing need to monitor water supplies to ensure they meet strict standards for human consumption. This often involves the testing of large volumes of water in order to detect relevant numbers of microbial contaminants which is time consuming and expensive. Automated methods and apparatus are being developed to assist in the large scale testing of samples for microbial contamination. The methods presently in use are often insensitive and do not allow the identification of specific microorganisms present in the samples being tested.

Flow cytometric detection of specific microorganisms relies on labelling the target organism with highly specific probes attached to fluorochrome molecules. To enable accurate detection, two or more different fluorescent labels need to be attached to the target organism (Vesey et al.1994A). The types of probes available for these techniques are monoclonal and polyclonal antibodies, lectins and oligonucleotides. The range of fluorochromes that can be coupled to these probes is limited. For example, many flow cytometers utilise a 488 nm laser to illuminate the sample, and accordingly, the choice of fluorochromes is limited to those which can be excited at 488 nm for those machines.

For a flow cytometer to distinguish one fluorochrome from another, the fluorochromes must emit at different wavelengths. There are only three types of fluorochromes presently available that excite at 488 nm and emit at wavelengths different enough to be distinguished by flow cytometry: green fluorochromes such as fluorescein isothiocyanate (FITC); red fluorochromes 06/03 '00 16:47 FAX 61 2 9810 8200 F.B. RICE CO. I]009 2 such as phycoerythrin (PE) and tandem fluorochromes. Unfortunately, the t~ndem fluorochromes are often not bright enough to be used in many applications. Therefore, flow cytometry is often limited to the detection of two fluorochromes. In applications such as the detection of specific microorganisms in a range of sample types, this poses a problem if there are only a small number of sites available for recognition on the microorganism.

The level of sensitivity that can b'e achieved with two fluorochromes is often not good enough for these applications.

The present inventors have developed methods of detecting 10 microorganisms in a fluid sample utilising particles and fluorescent labelled antibodies reactive to microorganisms.

Disclosure of the Invention Accordingly, the present invention consists in-a method of detecting :the presence and estimating the number of microorganisms of at least one predetermined type in a sample containing the microorganisms, the method comprising the steps of: treating the sample with at least one detectable fluorescent particle, each fluorescent particle bearing an antibody reactive to the microorganisms of the predetermined type, the sample being treated for a period of time 20 sufficient to allow the microorganisms of the predetermined type in the sample to bind to the fluorescent particle via the antibody; further treating the sample with an antibody labelled with a fluorescent marker having a different fluorescence spectrum to that of the fguorescent particle, the antibody being capable of binding to the microorganisms of the predetermined type and being the same antibody as used in step the sample being treated for a period of time sufficient to allow the antibody to bind to the microorganisms of the predetermined type; analysing the sample by flow cytometry so as to detect the presence of a fluorescent particle associated with one or more of the fluorescent markers, the antibodies being so selected that such an association is indicative of the presence of microorganisms of the predetermined type in the sample; and (c estimating the number of microorganisms-of the predetermined type in th sample by measuring the intensity of the fluorescence of the one or more flaorscent markers associated with the fluorescent particle.

In a preferred embodiment of the present invention the fluorescent p rticle is a fluorescent latex bead. The latex beads preferably have a 7 Ri 06/03 '00 16:47iFAX 61 2 9810 8200 F.B. !RICE CO. [010 S.y nominal diameter from 10 nanometres to 0.1 millimetres. The latex beads are preferably detectable by virtue of being fluorescently labelled. Mdre than one type of particl dan be used with each type bearing a antibody reactive to the same or differeit type of micioorgaism tobe detected.

r In a further peferred embodiment of the present invention the.

antibody is preferably a monoclonal antibody. The fluorescent markdts atached to the at least one antibody have different fluorescent spectra to that of the fluorescent particle. Using flow cytometry, the microorganisnis can be detected by the 10 presence of fluorescence of the labelled antibody or in combination with the size of the particle, or more preferably, fluorescence of both the marker and the particle. With regard to the d tection of the size of the particle, this icludes either detcting the knovn size of the particle or detecting or neasuring-for an increased size ca.sed by the tinding of microorganisms to 15 the particle.

In a still further preferred embodiment of the present invention the particle is labelled with several antibodies reactive to the same or different S. microorganisms. Furthermore, several different particles can also be used hiving the same or different antibodies bound thereto. For example, in step 20 several different antibodies reactive to the same or different microorganisms but provided with different fluorescent markers are utilised to allow the possible detection of more than one type of microorganism bound to the particle., SThe method according to the present invention preferably uses ne or more fluorescent markers that are excited at 488 nm and emit at wavelengths ranging from green t'infra-red. It will be appreciated by one skilled in the at that fluorescent markers that are excited at other wavelengths are also suitable for the present invention.; S The present invention is suitable for detecting multiple forms of the same species.of microorganism or detecting several different microorganisms from the same sample using one form of antibody for each type of nicroorganisms. The microorganisms bound to'the particle may be further treated or analysed after being detected by the method of the present in ention. The nunber of particles used in the present methods will depend on th type of particle, the type of sample being tested, and the number and type L7 06/03 '00 16:4 FAX 61 2 9810 8200 F.B. RICE CO. B011 Therefore, the number of particles should preferably be in excess to the of microoranisms in:the sample.i It will be appreciated that the nucroorganisms must come in contact with a particle to allow binding.

'therefore, the number of particles should preferably be in excess to te tumber' microorgaisms in a given sample to ensure detection of the microorganisms of interest. In order to assist in this regard usually atleast 3 particles per ml, preferably between 10 to I particles per ml are used.

When a sample has a lot of particilate material present then usually higher number of detectable particles is used in order to increase the possibility that the microorganisms present in the sample will come into contact with the 10 particles and bind. The present iivention has the advantage that the number of microorganisms of a given type in a sample dan also be estimated by adding a known number of detectable particles'to the sample and counting al of those particles to determine the number that have bound icroorganisms. Furthermore, by adding a known number of particles to the snmple it is also possible to confirm that the sample was correctly analysed by enumerating the number of particles detected.

In a preferred form step includes several different antibodies' each reactive to a different type of microorganisms, each different antibodyi-being labelled with a different fluorescent marker having a different fluorescence 20 spectrum to that of the fluorescent particle andfthe other labelled antibodies, such that several different predetermined types-of microorganisms are.

detected.

In an other preferred form the detectable fluorescent particle bears several different antibodies, each antibody being reactive to a different predetermined type of microorganisms, step includes the same several d iferent antibodies as present on the particle and each reactive to a different predetermined type of microorganisms, each different antibody being.

labelled with a different fluorescent marker having a different fluorestence spectrum to that of the fluorescent particle and the other labelled antibodies and being reactive to a different predeterrined type of microorganismns so as toallow detection of more than one predetermined type of microorganisms in the sample by analysing for the presence of more than one different fluorescent-labelled antibody associated with the fluorescent particle.; I R j/P* 0603 '0 6:SFAX 61 2 9810 8200.' F.B. RICE CO. j01 4/A The reset iventon i patiCularly suitable to detect. rcroorganisms vbhere usually one mhnatgni-rcgie y' antibodies generated threo Thmpeeatinvaienoscgnse theeto Te peseittinentrsh ve found that usually one main -antigen is presented by Czvptos~oyidium pjadthrfrtisognism is particual h6wever. that the method is not iiimited to the. detection of this microorganism.

In order that the nature of the present in'eto a emr lal understood, preferred forms thereof will be described with reference to the following examples and drawings'; Brief Descripotion of the Drawingas' SFigure 1 shows~flow cytomtry -scatter plo6ts representing Q ytospori diurn cocysts capturedc onto fluorescent beads and then boi4nd wi 'th a iFITC-conjupated- Czyptospdrid urn-specific antibody- and ft_ Figure 2 shows flow cytoinetiy scatter plots representing Adenotiius *captured onto fluorescent beads a'nd then bound with FITC-conjugated Adenovirus-specific antibody.

Modes for Carrying Out the Invention *MATERIALS AND MSETHODS 20- Coating, beads with -antibody ~..TransFluorSphere 488/685 latex beads were coated as recommended by Molecular Probes (Eugene, USA) With antibody"specific to the microorganism 0f interest.

SAntibody (2 -g specific to' either: Giyptosporidlum (Biox, Sydney), Adenovirus (Silenus,: Melbourne) or SaLmonella typhimurium (Wellcome Diagnostics) was dissolved in 1 ml of 50 mM Tris buffer (pH 8.4) and then dialysed overnight at 'C 0 against 50 mM MVES buffer (pH The antibody was then mnixed with 5 ml of 0.2%/6 (wlv) I pin latex beads (TransFluor~phere WO 96/31777 PCT/AU96/00192 488/685, Molecular Probes, Eugene USA) and incubated at room temperature for 15 min before the addition of 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (40 mg). The pH was then adjusted to 6.5 by the addition of 0.1 M NaOH. After incubation at room temperature for 2 hours, glycine was added to give a final concentration of 100 mM and the sample incubated for a further 30 min. The beads were then pelleted by centrifuging (13000g for 2 min) and the pellet resuspended in 1% bovine serum albumin (BSA) in phosphate buffered saline (pH The washing procedure was repeated three times before the addition of sodium azide. The final sample volume was 4 ml. Beads were sonicated for 30 min prior to use.

Using beads to label Cryptosporidium oocysts in water samples River water samples were concentrated by calcium flocculation (Vesey et al. 1993). Portions (1 ml) of the concentrate were seeded with approximately 1,000 oocysts. BSA was added to a concentration of 1% (w/v) prior to the addition of 20 pl of the crypto-antibody-coated bead suspension.

Samples were then incubated at room temperature on a rotary mixer for min.

Oocysts were then labelled with a second fluorochrome. Monoclonal antibody, specific to Cryptosporidium oocysts walls, conjugated with fluorescein isothiocyanate (Cellabs Pty Ltd, Sydney, Australia) was added ml) and the samples incubated at 37 0 C for 20 min.

Using beads to label Salmonella Salmonella typhimurium was cultured on MacConkey agar, fixed in formalin for 15 min and washed by centrifuging (13000g for 10 min) and resuspending in PBS. An aliquot (100 pl) containing approximately 1 x 106 cells was mixed with 20 l1 of bead suspension (coated with Salmonella antibody) and then incubated on a rotary shaker for 30 min a room temperature. Salmonella cells attached to beads were labelled with a second fluorochrome by incubating with rabbit anti-Salmonella antibody (Wellcome Diagnostics), washing by centrifuging at 13000g for 30 seconds, resuspending in a goat anti-rabbit 7-amino-4-methylcoumarin-3-acetic acid (AMCA) (Dako, Glustop, Denmark) conjugated antibody and incubating for min at 37 0 C. Samples were examined using epifluorescence microscopy.

Samples were analysed immediately.

WO 96/31777 PCT/AU96/00192 6 Using beads to label Adenovirus Adenovirus was cultured in human epithelial cells, harvested by freeze thawing the cells and then purified from cell debris by centrifuging (13000g for 2 min) and retaining the supernatant. The supernatant was then mixed with 20 pl of the bead suspension (coated with Adenovirus antibody) and then incubated on a rotary shaker for 30 min at room temperature.

Adenovirus attached to beads were labelled with a second fluorochrome by incubating with the same Adenovirus antibody conjugated with FITC for min at 37 0

C.

Sample analysis Samples were analysed using a Becton Dickinson Facscan flow cytometer. The discriminator was set on red fluorescence (FL4) at a level slightly less than the fluorescence of the beads. A region (R1) on a scatter plot of green fluorescence (FL1) verses side scatter (graph 1) was defined which enclosed the FITC-labelled oocysts. This region was then used to gate a scatter plot of red fluorescence verses side scatter (graph A region was defined on this second scatter plot which enclosed oocysts attached to beads. The same process was also used for analysis of Adenovirus (Fig. 2).

Colour compensation was performed to separate the fluorescence of the beads from the fluorescence of the labelled organism. Red fluorescence was progressively subtracted from green fluorescence until a second population appeared on the green fluorescence verses side scatter graph.

RESULTS

Cryptosporidium Analysis of the CrOptosporidium sample by flow cytometry resulted in a distinct population on graph 1 (Figure This population represents all beads and was enclosed within a region Gating a graph of side scatter verses FITC on the region Rl produced the scatter graph 2 (Figure Two populations are observed on graph 2, a large population with a low green signal (spill over from high level of red fluorescence from the beads) which represent the beads and a smaller population with a high FITC signal which represent beads attached to FITC-labelled oocysts.

Adenovirus Viruses could be detected on the flow cytometer when using the gating and colour compensation procedures that were used for Cryptosporidium. A scatter plot representing a large population of beads WO 96/31777 PCT/AU96/00192 7 with low FITC fluorescence and a small population of beads with high FITC fluorescence (Figure 2) was observed. This second population represents viruses attached to beads and labelled with FITC. The negative control did not contain any beads with a high FITC signal.

Salmonella Examination of the beads using epifluorescence microscopy revealed red fluorescing beads attached to blue (AMCA) fluorescing Salmonella cells.

The detection of specific or predetermined microorganisms with flow cytometry has the potential to replace existing methodologies for the detection of microorganisms in samples ranging from clinical fluids, water, food and beverages. On way to enable simple and rapid flow cytometric detection of low numbers of microorganisms is to use at least two different coloured fluorochromes for attached to the microorganisms. These fluorochromes are attached to the microorganism via highly specified ligands such as antibodies. This has been achieved previously by conjugating different coloured fluorochromes directly to antibodies (Vesey et al.1994A).

The present inventors have shown that microorganisms can be detected by flow cytometer by attaching fluorescent beads to the microorganisms. The population representing oocysts attached to beads displayed in graph 2 (Figure 1) is an identifiable population totally clear from any unassociated coloured bead or interfering noise. The population representing viruses attached to beads in Fig. 2 is also an identifiable population.

The technique of using a fluorescent particle to tag a specific or predetermined microorganism with a fluorescent label has several advantages over using only a fluorochrome-conjugated antibody. Firstly, only a single bead needs to be attached to the microorganism to achieve detectable fluorescence. To achieve the same level detection using only a fluorochrome conjugated antibody requires thousands of antibodies to be attached to the microorganism. These thousands of antibodies cover and mask available antigen sites. If only a single type of antigen is available on the surface of a microorganism, then it is not always possible to label the surface of the organism with a second antibody. The present inventors have found that only one antigen is presented by Cryptosporidium and therefore this organism is difficult to detect by previous methods.

WO 96/31777 PCT/AU96/00192 8 If the microorganism is labelled with an antibody coated fluorescent bead then there are many antigen sites still available on the surface of the organism for attaching an antibody conjugated to a fluorochrome. This technique enables two colour fluorescence labelling of a microorganism with a single antibody as shown by the present inventors.

A further advantage of the fluorescent bead labelling technique is that it enables the use of new fluorescence emission wavelengths. Until recently the number of different colours that can be detected by a single laser flow cytometer has been limited to two. These are a green fluorochrome such as FITC and a red fluorochrome such as PE. A third colour is now possible using tandem fluorochromnes such as PE/Texas red where the PE pumps the Texas red. These tandem fluorochromes however are not bright enough for many applications. When attempting to detect microorganisms the fluorescence signals need to be very bright. The fluorescent beads enable the use of a third and even a fourth very bright fluorescent signal. This is because beads with a range of different fluorescent emissions are available. Beads with emissions as far into the infra-red as 720 nm are available.

Examples of fluorescent beads and their production that are suitable for use in the present invention can be found in US patent 5326692.

The use of fluorescent beads as a label improves flow cytometric detection. This is because the beads can be used as the size or fluorescence discriminator. The cytometer can be set so that it ignores all other particles except for the beads. This overcomes coincidence problems due to the sample containing more particles than the cytometer can examine. It also means that a known number of particles need to be examined for all samples.

The bead technology is highly applicable to the detection of bacteria.

It enables multiple bright fluorescence signals to be achieved on the surface of a range of bacteria. The production of antibodies to large groups of bacteria (eg all gram negative bacteria) and then coating beads with these antibodies will allow the use of a single reagent for a range of microorganisms or their sub-types.

The application where this bead technology will have the most benefits will be the flow cytometric detection of very small particles such as viruses. Coating beads with virus specific antibodies and reacting with WO 96/31777 PCT/AU96/00192 9 samples captures viruses onto the beads. The virus is then labelled with a second fluorochrome enabling detection. This is the first, simple virus detection procedure that can be performed within minutes.

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

References Vesey, Narai, Ashbolt, Williams, K.L. and Veal, D. 1994A.

Detection of specific microorganisms in environmental samples using flow cytometry, p.489-522. In Methods in Cell Biology-Flow Cytometly Second Edition. Academic Press Inc., New York.

Vesey, Hutton, Champion, Ashbolt, Williams, Warton, A. and Veal, D.A. 1994B. Application of flow cytometric methods for the routine detection of Cryptosporidium and Giardia in water.

Cytometry, 16: 1-6.

Vesey, Slade, Byrne, Shepherd, and Fricker, C.R., 1993. A new method for the concentration of Crptosporidium oocysts from water. J. Appl. Bact. 75,82-86.

Claims (11)

1. A method of detecting the presence and estimating the number of microorganisms of at least one predetermined type in a sample containing the microorganisms, the method comprising the steps of: treating the sample with at least one detectable fluorescent particle, each fluorescent particle bearing an antibody reactive to the microorganisms of the predetermined type, the sample being treated for a period of time sufficient to allow the microorganisms of the predetermined type in the o sample to bind to the fluorescent particle via the antibody; 10 further treating the sample with an antibody labelled with a fluorescent marker having a different fluorescence spectrum to that of the fluorescent particle, the antibody;being capable of binding to the microorganisms of the predetermined type and being the same antibody as used in step the sample being treated for a period of time sufficient to allow the antibody to bind to the microorganisms of the predetermined type; Ic) analysing the sample by flow cytometry so as to detect the presence of a fluorescent particle associated with one or more of the fluorescent markers, the antibodies being so selected that such an association is indicative of the presence of microorganisms of the predetermined type in the sample; and 20 estimating the number of microorganisms of the predetermined type in the sample by measuring the intensity of the fluorescence of the one or more fluorescent markers associated with the fluorescent particle.

2. The method according to claim 1 wherein the fluorescent particle is a fluorescent latex bead.

3. The method according to claim 2 wherein the fluorescent latex bead has a nominal diameter from 10 nanometres to 0.1 millimetres.

4. The method according to any one of claims 1 to 3 wherein the sample is treated with at least 10 3 detectable fluorescent particles per millilitre sample. 5, The method according to claim 4 wherein the sample is treated with between 104 and 107 detectable fluorescent particles per millilitre sample.

6. The method according to anyone of claims 1 to 5 wherein the microorganisms are selected from the group consisting of protozoa, bacteria, fungi and viruses. 'v u T -J l' 06/03 '00 16:46 FAX 61 2 9810 8200 F.B. RICE CO. 007 11

7. The method according to claim 6 wherein the microorganisms are viruses.

8. The method according to any one of claims 1 to 7 wherein the antibody is a monoclonal antibody.

9. The method according to any one of claims 1 to 8 wherein the one or more fluorescent markers are excited at 488 nm and emit at wavelengths ranging from green to infra-red. The method according to any one of claims 1 to 9 wherein the microorganisms are detected by the presence of fluorescence of the labelled 10 antibody associated with a particle, by fluorescence of the labelled antibody in combination with the size of the fluorescent particle, or by fluorescence of both the antibody and the fluorescent particle.

11. The method according to any one of claims 1 to 10 wherein the fluorescent particle bears several different antibodies each reactive to a different predetermined type of microorganisms.

12. The method according to any one of claims 1 to 11 wherein step (b) includes several different antibodies each reactive to a different type of microorganisms, each different antibody being labelled with a different fluorescent marker having a different fluorescence spectrum to that of the 20 fluorescent particle and the other labelled antibodies, such that several different predetermined types of microorganisms are detected. 3 The method according to any one of claims 1 to 11 wherein the' detectable fluorescent particle bears several different antibodies, each antibody being reactive to a different predetermined type of microorganisms, step includes the same several, different antibodies as present on the particle and each reactive to a different predetermined type of microorganisms, each different antibody being labelled with a different fluorescent marker having a different fluorescence spectrum to that of the fluorescent particle and the other labelled antibodies and being reactive to a different predetermined type of microorganisms so as to allow detection of more than one predetermined type of microorganisms in the sample by aqalysing for the presence of more than one different fluorescent-labelled a qtibody associated with the fluorescent particle, r 4' u uL 0ij 06/03 '00 16:47,' FA 61 2 9810 8200 F.B. RICE CO. 110O8 12 I

14. The method according to apay one of claiMs 1 to 1.3 wherein the, number of microorganisms of a pieerniindte inP tesmple is estimated by adding to the sample -a known number of detectable flubres cent Particles and analysn all the fluorescent particles- in the- sample for, dsociation with one;or more of the fluorescent markers and estimating the n~umber of microorganisns inthe ample from~ithe number of detectable forescent particles -associate with the fluore~icent markers. Dated this sixth day of March 2000 V4 MACQUARIE RESEARCH LTD, AUSTRALIAN WATER a TECHNOLOGIES PTY LTD ~Patent Attorneys for the A piat F BRICE CO W L LU