WO1991006674A1

WO1991006674A1 - Method and device for the detection of antibiotic resistance

Info

Publication number: WO1991006674A1
Application number: PCT/GB1990/001697
Authority: WO
Inventors: William Joseph Harris; Francis Joseph Carr
Original assignee: Scotgen Ltd.
Priority date: 1989-11-06
Filing date: 1990-11-06
Publication date: 1991-05-16
Also published as: GB8924989D0

Abstract

A method for the quick and easy detection of antibiotic resistance in a sample and a kit that incorporates this method. The method involves providing a support; a first probe capable of specifically binding to part of the nucleic acid encoding the antibiotic resistance, and which is immobilisable on the support before and during the test procedure; a second probe capable of specifically binding another part of the nucleic acid encoding the antibiotic resistance, this second probe being labelled so as to be capable of producing a signal; allowing nucleic acid from the sample to come into contact with the first probe and the labelled second probe; detecting any signal from the labelled second probe bound to the support by cohybridisation of the immobilised first probe and the labelled probe to antibiotic resistance nucleic acid.

Description

METHOD AND DEVICE FOR THE DETECTION OF ANTIBIOTIC RESISTANCE Technical Field

The invention relates to a method of detecting antibiotic resistance, in particular in bacteria; to a kit for use in such a method; and to components usable in such a method. Background Art Antibiotics may be used in the treatment of infections caused by, for example, pathogenic bacteria. Examples of such infections are urinary tract infections (UTI). The incidence of UTIs worldwide is second only to upper respiratory tract infections although the frequency of testing of UTI far exceeds that for respiratory pathogens. In practice, the major activity of clinical bacteriology laboratories is in testing urine samples for the presence of bacterial pathogens and, subsequently, for the resistance of those pathogens bacteria to antibiotics, thus guiding the clinician in the choice of antibiotics for UTI therapy.

The choice for an antibiotic may be impeded by bacterial resistance to the most commonly used antibiotics. A common feature of bacterial resistance to a wide range of antibiotics is that resistance is effected by small circles of self replicating DNA called plasmids. The ready transfer of such plasmids between bacteria accounts for the rapid speed of drug resistance through environmental bacteria therebynecessitating before treatment a screen of a range of different antibiotics (Table 1). Table I :

Mechanism of Plasmid

Borne Resistance

Amoxycillinl Penicillins Ampicillin j

Cleavage of lactam ring (beta lactamases)

Cephradine )

Cephalosporins

Cephuroxime j Gentamicin N-acetylation, O-

Aminoglycosides phosphorylation (transferases)

Spectinomycin Tetracycline altered antibiotic transport

Trimethoprin altered target enzyme (dihydrofolate reductase)

Ciprofloxacin

Quinolones altered DNA gyrase Cafotaxime (chromosomal)

During the last decade, a variety of new rapid tests have been introduced which provide same-day screening of urine samples for significant bacterial infection. E.coli is the organism predominantly isolated from such samples, and has been reported in over 80% of cases of significant bacteriuria (Mond et al ig65; Lancet, i., 514-416; Trepeta and Edberg, ig84, J Clin Microbial 1 No 2, 172-174). Many new methods of detection are based upon the detection of enzymes characteristic for the bacteria, in particular detection of the enzymes β -galactosidase and f? - glucouronidase using chromogenic substrates (Kilian and Buloe, 1976; Acta Pathol Microbial Scan, Sect B 84, 245-251, 1979, Acta Microbial Scand Sect B {3 271-276; Hansen and Yourassowsky, J. Clin Microbial 1984, 20_, No 6 1177-1199). Such tests are progressively circumventing the need for the standard overnight culture screen, and through testing by GPs, such tests are reducing the work load of clinical bacteriology laboratories. However, for the rational administration of antibodies to UTI patients, culture of bacteria on antibiotic plates remains the only reliable means for assessing profiles of antibiotic resistance.

It is therefore desirable to provide an improved method for determining antibiotic resistance which is easy to perform, specific and gives a fast result. It should also be an alternative to the widely used culture methods. Disclosure of Invention

According to the present invention there is provided a method for detecting antibiotic resistance in a sample which comprises providing a support, a first probe capable of specifically binding to part of the nucleic acid encoding the antibiotic resistance, means being provided whereby the probe is immobilisable on said support, and a second probe capable of specifically binding another part of the nucleic acid encoding the antibiotic resistance, the second probe being labelled so as to be capable of producing a signal; allowing nucleic acid from the sample to come into contact with the first probe and the labelled second probe; immobilising the first probe on the support; and detecting any signal from labelled second probe bound to the support by cohybridisation of the immobilised first probe and the labelled probe to antibiotic resistance nucleic acid.

The probes may be oligonucleotide probes, isolated gene segments or corresponding RNA. The nucleic acid encoding resistance may be RNA or DNA. The first probe may be immobilised on the support before it interacts with the target nucleic acid. Alternatively the immobilisation may happen during and/or after such interaction.

In one embodiment of the invention a ligand-tagged probe can be immobilised on a dipstick via a ligand binding moiety, for example an antibody (Ab) to the ligand, during the test procedure. Thus an oligonucleotide may have a non-nucleic acid ligand group attached. The ligand of the ligand tagged probe may be, for example, BUdR, (bromodeoxy uridine), FITC (fluorescein isothiocyanate), nitroindophenol, dinitrophenol or penicillin. BUdR can be attached in a manner conventional in nucleic acid synthesis, e.g. using a phosphor amidite derivative. FITC and nitroindophenol can be incorporated by reaction of a succimidyl derivative with a 5' amino group on the oligonucleotide. Dinitrophenol can be attached via dinitrobenzene sulphonic acid. Penicillin can be attached via penicillinic acid. Preferably the DNA probes are specific to genes encoding resistance to: penicillins/cephalosporins; trimethoprim; aminoglycosides; or tetracycline.

The method is suitable for the detection of bacterial antibiotic resistance in a clinical sample, such as urine. Bacterial DNA from the sample is suitably prepared by lysing the bacteria with an alkaline buffer and denaturing the DNA. The labelled probe may then be added with a neutralising buffer.

The label may be an enzyme, and a suitable signal producing system is one which produces a colour in the presence of the product resulting from the action on the enzyme on a suitable substrate.

Preferably, the method provides for the simultaneous detection of different classes of antibiotic resistances by providing distinct regions of a first set of probes on the support and a second set of labelled probes which are specific for antibiotic resistance genes of each of the classes.

The support may be in the form of a dipstick to which said first set of probes are attached in separate discrete areas. The dipstick may be made of paper, nylon, plastics or any other suitable material. Alternatively, the support may be in the form of a multiwell microtitration plate, with probes of the first set immobilised in separate wells.

The invention will now be described, by way of example only and with reference to the accompanying^•drawings. Brief Description of Drawings

Figure 1 shows diagrammatically a dipstick with four regions for detecting antibiotic resistance, and

Figure 2 shows diagrammatically a multiwell format alternative to the dipstick of Figure 1. Best Modes for Carrying Out the Invention

Referring to Figure 1 there is shown a dipstick 10, for example of plastic, nylon, paper or other suitable material, having thereon four separate strips A-D. Each of the strips comprises a DNA probe, for example an oligonucleotide probe, specific for genes conferring resistance to different antibiotics: strip A - penicillin strip B - trimethoprim strip C - aminoglycosides strip D - tetracycline

Each DNA probe is attached to the dipstick by conventional procedures, such as chemical covalent linkage of the 3' -OH end, or through ligand-mediated processes, or any other suitable process.

In use, for the determination of the antibiotic resistance of bacteria in a urine sample, a small volume of urine is treated with an alkaline buffer to lyse bacteria and denature the DNA therein. Then a neutralising buffer is added which contains a set of DNA probes, e.g. oligonucleotide probes, specific for each of the four antibiotic resistance genes, these probes being labelled with enzyme, e.g. alkaline phosphatase. Following a short period of hybridisation, during which the probes hybridise to any corresponding antibiotic 5 resistance gene from the sample, the dipstick is briefly

* introduced to bind the now labelled bacterial DNA. Finally, the dipstick is washed and introduced into a substrate for the enzyme which is part of a signal producing system, leading to colour development. The result of such a sandwich DNA probe 10 hybridisation assay is a profile of antibiotic resistance, for example, as depicted in Fig. 1. Figure 1 indicates, by way of example, resistance to penicillin and aminoglycosides, but not to trimethoprim or tetracycline.

It will be appreciated that the lengths of the probes are 15 chosen so as to provide sufficient specificity and stability during hybridisation. Also the DNA of each probe immobilised on the dipstick should hybridise to a different region of the antibiotic resistance gene from the corresponding labelled probe, so that both can simultaneously hybridise to the 20 bacterial DNA.

Referring to Figure 2, there is shown a multiwell plate 12, for example a conventional 12 x 8 well microtitre plate, in which the first set of DNA probes are attached to separate wells or groups of wells A-D by conventional methods.

I 25 Urine samples, treated and exposed to a set of labelled probes as described above, are distributed between the wells of the plate, and after hybridisation the wells are washed before the addition of enzyme substrate for colour development. The result will be a profile of antibiotic resistance, for example as depicted in Figure 2. As Fig. 2 indicates, using a multiwell assay it is possible to test several different test samples at the same time by introducing them in rows on the plate.

It will be appreciated that instead of DNA probes, RNA probes could be used. The number of different antibiotic resistances tested may also vary.

The DNA sequences of many bacterial antibiotic resistance genes have now been sequenced, and no doubt others will be sequenced in due course, and their use is included within the scope of this invention. Although only the testing of a clinical sample has been described, other samples that may contain bacteria with antibiotic resistance which may be tested, are, for example recombinant samples.

A practical example will now be described in more detail.

Example

This example describes the preparation of dipsticks and their use to detect and discriminate between two plasmid DNA sequences encoding resistance to the antibiotics tetracycline and kanamycin. In this example, both probes are initially in solution but one beomes immobilised by interaction with an antibody associated with a dipstick. Unless otherwise stated, all antibodies, plasmids and other reagents were obtained from Sigma Chemical Co., Poole, Dorset, England. ^{5 1}* Purification of antibodies

Monoclonal antibodies to BuDR (bromodeoxyuridine) were obtained from Amersham International. Where necessary, antibodies were further purified by high pressure liquid affinity chromotography using a column of immobilised 10 streptococcal protein G.

2. Preparation of dipsticks

Sheets of nitrocellulose membrane (Sartorius), pore size 0.45μm, were prewetted with tris/saline (lOmM tris, 0.9% (w/v) sodium chloride, pH7.4) and placed in a "Bio-Slot" apparatus 15 (BioRad). A ligand binding moiety "was provided in the form of an antibody to BuDR. Thus, lμg of purified antibody in 200μl tris/saline was placed in each well and allowed to pass through the membrane. After half an hour, the wells were washed with 200ul tris/saline. The membrane was removed and 20 stored at 4°C in tris/saline containing 5% (w/v) bovine serum albumin (BSA).

3. Preparation of oligonucleotide probes

Oligonucleotides were synthesized on an Applied Biosystems model 381A DNA synthesizer according to the manufacturer's , 25 instructions, using the phosphoramidite system with reagents by Cruachem (Glasgow, Scotland). "Ligand tagged probes", i.e. probes labelled with BUdR (bromodeoxyuridine), were prepared using the phosphoramidite derivative of BUdR. This was incorporated as the 5'-most base of the oligonucleotide. For probes which were to be labelled with FITC (fluorescein isothiocyanate) or alkaline phosphatase, a modified base with a free primary amino group at the end of a 6-carbon spacer arm was attached as the 5'-most base. Oligonucleotides were used without further purificatio .

4. Labelling of capture probes with FITC As an alternative to BUdR as ligand, probes can be labelled with FITC as follows. 20μg of 5'-amino-oligonucleotide was mixed with 20μl FITC (4mg/ml in N,N'-dimethylformamide), 30μl of IM carbonate/bicarbonate buffer, pH9.0, and distilled water to a final volume of 150μl. This reaction mixture was incubated overnight at room temperature in the dark, then extracted five times with ethyl acetate to remove free FITC. The labelled probe was precipitated by the addition of 1/10 volume of 3M sodium acetate, pH5.2, and 2 volumes of ethanol, followed by incubation for 2 hrs at -20C. The pellet was recovered by centrifugation (15 minutes in a microcentrifuge). The supernatant was decanted off and the pellet dried under vacuum. The labelled probe was redissolved in 0.2M carbonate/bicarbonate buffer, pHg.0.

5. Labelling of reporter probes with alkaline phosphatase 3.5 nmoles of 5'-amino-oligonucleotide in 0.1M sodium bicarbonate, 2mM EDTA, was added to 50μl of 10 mg/ml disuccinimidyl suberate (Pierce Chemical Co) in dimethyl sulphoxide, incubated for 5 minutes at room temperature in the dark, dialysed against several changes of O.IM sodium phosphate, pH6.0, overnight at room temperature, and the dialysed material was dried under vacuum. The dried material was rehydrated with 200μl O.IM carbonate/bicarbonate, pH8.25, containing 3M sodium chloride, 0.05% (w/v) sodium azide and 1 mg alkaline phosphatase. After 16 hours at room temperature, the reaction products were separated by gel filtration using a Zorbax GF250 column equilibrated with 0.2M sodium phosphate, pH7.0. The conjugated reporter probe is the first peak to emerge.

6. Test procedure

Strips (5cm x 1cm) cut from the membranes produced as described in section 2 above were prehybridized for 1 hour at 37C in 5 ml of 4XSSC (0.6M sodium chloride, 0.06M sodium citrate, pH7.0), 10% (v/v) deionized formamide, 5% (w/v) BSA and 0.1 mg/ml heat-denatured sheared salmon sperm DNA. At the same time plasmids pBR322 and pUBHO (O.lμg) were incubated for 1 hour under the same conditions in the presence of appropriate reporter and ligand probes (10-100 ng/ml) (the ligand probes were BuDR-labelled) . The dipsticks were then immersed in this hybridisation mixture and incubation continued for a further hour. The dipsticks were then removed, washed 4 times for 5 minutes each in 4XSSC, then immersed in 0.1M tris, 0.1M sodium chloride, 5mM magesium chloride pHg.5 containing 0.3 mg/ml NBT and 0.2 mg/ml BCIP. Colour developed within 30 minutes. The dipsticks were then removed from the substrate solution and washed with water.

The results show specific detection of the tetracycline resistance gene of pBR322 by probes directed against this gene, and similar detection of the kanamycin resistance gene of pUBHO.

Claims

1. A method for detecting antibiotic resistance in a sample which comprises providing a support, a first probe capable of specifically binding to part of the nucleic acid encoding the antibiotic resistance, means being provided whereby the probe is immobilisable on said support, and a second probe capable of specifically binding another part of the nucleic acid encoding the antibiotic resistance, the second probe being labelled so as to be capable of producing a signal; allowing nucleic acid from the sample to come into contact with the first probe and the labelled second probe; immobilising the first probe on the support; and detecting any signal from labelled second probe bound to the support by cohybridisation of the immobilised first probe and the labelled probe to antibiotic resistance nucleic acid.

2. A method according to claim 1 wherein said step of immobilising the first probe is performed prior to said step of allowing sample nucleic acid to come in contact with the probes.

3. A method according to claim 1 wherein said step of immobilising the first probe is performed at the same time as and/or after said step of allowing nucleic acid to come in contact with the probes. ^'7 4

4. A method according to claim 1, 2 or 3 wherein said step of providing a support, first probe and immobilisation means comprises tagging a nucleic acid probe with a ligand, and providing a support with a complementary ligand-binding moiety.

5. A method according to claim 4 wherein the ligand of the ligand-tagged probe is a bromodeoxyuridine is (BUdR), fluorescein isothiocyanate (FITC), nitroindophenol, dinitrophenol or penicillin moiety. 6. A method according to claim 43 wherein the ligand-binding moiety comprises an antibody.

7. A method according to any preceding claim wherein said first and second probes are complementary to DNA or RNA sequences of a target microorganism which confer antibiotic resistance on the target microorganism.

8. A method according to any of the preceding claims wherein the first and second probes are capable of specific binding to genes encoding a class of resistance selected from resistance to: penicillins/cephalosporins; trimethoprim; aminoglycosides; and tetracyline. g. A method according to any of the preceding claims wherein the sample is urine.

10. A method according to any of the preceding claims wherein the sample is bacterial and is prepared by lysing the bacteria with an alkaline buffer and denaturing the DNA. 7 5

11. A method according to any one of the preceding claims wherein the label of said second probe comprises an enzyme, and said step of detecting any signal from labelled second probe involves the use of a signal producing system for producing a colour in the presence of a product resulting from the action of the enzyme on a substrate.

12. A method according to any one of the preceding claims in which provision is made for the simultaneous detection of different classes of antibiotic resistances by providing distinct regions of members of a set of said first probes on the support, and providing a set of said second labelled probes, each of said sets having members which are specific for antibiotic resistance genes of each of the classes.

13. A kit for use in performing a method for detecting antibiotic resistance in a sample, which method comprises providing a support, a first probe capable of specifically binding to part of the nucleic acid encoding the antibiotic resistance, means being provided whereby the probe is immobilisable on said support, and a second probe capable of specifically binding another part of the nucleic acid enclosing the antibiotic resistance, the second probe being labelled so as to be capable of producing a signal; allowing nucleic acid from the sample to come into contact with the first probe and the labelled second probe; immobilising the first probe on the support; and detecting any signal from labelled second probe bound to the support by cohybridisation of the immobilised first probe and the labelled probe to antibiotic resistance nucleic acid, said kit comprising said first and second probes and said support.

14. A kit according to claim 13 wherein said kit permits the simultaneous detection of different classes of antibiotic resistances, and comprises a set of said first probes and a set of said second probes, each of said sets having members which are specific for antibiotic resistance genes of each of the classes; said set of first probes being immobilised on said support so as to provide distinct regions corresponding to different classes.

15. A kit according to claim 14 wherein the support for the immobilised probes is in the form of a dipstick to which said first set of probes are attached in separate discrete areas. 16. A kit according to claim 15 wherein said dipstick comprises a substrate of paper, nitrocellulose membrane, or nylon.

17. A kit according to claim 14 wherein the support is in the form of a multiwell microtitration plate, with probes of the first set immobilised in separate wells.

18. A kit according to claim 13 for use in a method T 7

according to any of claims 1-12.

19. A support for use in detecting antibiotic resistance in a sample, said support having a plurality of distinct regions each of which has immobilised to it a respective probe capable of selectively binding to nucleic acid encoding antibiotic resistance of a respective class.

20. A probe comprising an oligonucleotide capable of hybridising to a gene encoding antibiotic resistance; said oligonucleotide being coupled to a ligand for use in immobilising the probe to a support.

21. A probe according to claim 20 wherein said ligand is a bromodeoxyuridine moiety.

22. In combination, a probe according to claim 20 and a support having complementary means for binding said ligand thereby to immobilise said probe.