WO2004044187A1 - Rapid detection of bacterial spores - Google Patents
Rapid detection of bacterial spores Download PDFInfo
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- WO2004044187A1 WO2004044187A1 PCT/GB2003/004923 GB0304923W WO2004044187A1 WO 2004044187 A1 WO2004044187 A1 WO 2004044187A1 GB 0304923 W GB0304923 W GB 0304923W WO 2004044187 A1 WO2004044187 A1 WO 2004044187A1
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- Prior art keywords
- sample
- bacterium
- bacteria
- autoinducer
- cultured
- Prior art date
Links
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/32—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Bacillus (G)
Definitions
- the present invention is concerned with methods for the rapid detection of the presence of bacterial spores in a sample and with the identification of the bacterial spores.
- Bacillus anthracis is an aerobic, non-motile Gram-positive rod. Most members of the genus Bacillus are saprophytic, and are present in soil, water, air and vegetation. B. cereus is an important cause of food poisoning, but B. anthracis is the most virulent and best- known pathogen. Its virulence is based on two plasmids, pXOl which codes for the protective antigen, oedema factor and lethal factor, that comprise anthrax toxin, and pX02 which encodes the protective capsule.
- B. anthracis form heat-resistant spores that can survive for many years in soil and ground water, and it is this property that makes B. anthracis a particular focus for the development of biological weapons.
- other spore-forming bacteria include those of the genus Clostridium. This genus contains a number of highly pathogenic species, including C. perfringens (the cause of gas gangrene), C. tetani (tetanus) and C. botulinum (botulism).
- Anthrax affects herbivores such as sheep, goats, cattle and horses. Bacteria are excreted in the faeces and urine of infected animals, and may form spores in the environment. Human infection may occur by contact with infected animals or contaminated animal products (hides, wool, bone meal, etc.), or by inhalation of spores.
- the bacteria multiply and produce anthrax toxin, leading to oedema.
- anthrax is rare in developed countries and when it does occur it is relatively easily diagnosed by typical culture on blood agar plates and other selective nutrient media, and it can be successfully treated by early administration of high doses of penicillin or ciprofioxacin.
- B. anthracis spores might be deliberately and maliciously dispersed, however, there is an urgent need for rapid and sensitive techniques for environmental testing for B. anthracis spores, in order to pre-empt widespread human infection.
- N-acyl homoserine lactones which are composed of derivatives of amino acid and fatty acid molecules. This family of molecules plays a key role in the mechanisms by which Gram-negative bacteria monitor population densities, factors that are important in the virulence of a number species.
- a purported bacterial autoinducer was also isolated by Lyte, M., et al. (FEMS Microbiol. Lett. 1996 Jun 1; 139(2-3): 155-9, PMID: 8674983) having a molecular weight of approximately 10,000 Da (see also Lyte, M.J., Endocrinol. 1993 Jun;137(3):343-5, PMID: 8371072; US 5629349).
- WO 98/53047 states that the autoinducer may be used to assay viable but non-culturable bacteria, no mention is made of culturing bacterial spores using the autoinducer.
- the present invention provides a method to do this and seeks to overcome the prior art disadvantages.
- a method for culturing bacterial spores in a sample comprising the steps of: (a) contacting said sample with a bacterial growth autoinducer, said bacterial growth autoinducer being characterised in that it has the following properties:
- MgS0 4 at pH 7.5 supplemented with 30% bovine serum, vol/vol); (ii) it is stable for 45 minutes at 100 °C; (iii) it is stable to lyophilisation; (iv) it has a negative charge and binds to an anion exchange resin;
- method step (a) may be preceded by the step of treating said sample to inactivate micro-organisms (for example to kill them) contained therein other than bacterial spores.
- the treatment may be to inactivate bacteria other than those in spore form.
- the inactivation treatment may for example kill bacteria such as Bacillus species not in spore form and other micro-organisms including fungi.
- the inactivation treatment may be anything which does not inactivate bacterial spores but which does inactivate other chosen micro-organisms. It may for example be a heat- treatment which inactivates bacteria not in spore form - a sample can for example be heated to 70 °C for a period of at least 5 minutes, for example at least 10, 15, 20, 25 or 30 minutes. Alternatively, an alcohol such as iso-propanol can be used, as can disinfectants and other techniques as appropriate such as sonication.
- the bacterial growth autoinducer may further be characterised in having at least one of the following characteristics: (a) it is stable in prolonged storage in a dried state and/or in solution; and
- the bacterial growth autoinducer may further be characterised in having at least one of the following characteristics: (a) it can be produced by bacteria grown in Luria broth, tryptone soya broth, M9 minimal medium and Davis-Mingioli minimal medium as well as by the same bacteria grown in serum SAPI medium;
- bacterial growth autoinducers which can be used in the methods of the present invention include those isolated from Escherichia coli, Yersinia ruckeri, Hafnia alvei, and Salmonella enterica serovars Enteriditis and Typhimurium.
- the autoinducers are typically not species-specific, instead an autoinducer from one species being promiscuous and often being able to induce the growth of bacteria from a range of other species.
- the germination and culturing of B. anthracis spores is induced by the autoinducer.
- the present invention is not limited to the use of autoinducers from the above list of species alone, but may include other species of the family Enter obacteriaceae.
- Culturing may be performed using conventional culture media in addition to the bacterial autoinducer.
- a method for the detection of bacterial spores in a sample comprising the steps of: (a) treating said sample to inactivate bacteria contained in it which are not in spore-form; (b) contacting said treated sample with a bacterial growth autoinducer, said bacterial growth autoinducer being characterised in that it has the following properties: (i) it can be produced by a Gram-negative bacterium in response to noradrenaline in serum SAPI medium (2.77 mM glucose, 6.25 mM ⁇ H 4 ⁇ 0 35 1.84 mM KH 2 P0 4 , 3.35 mM KCl, 1.01 mM MgS0 4 at pH 7.5 supplemented with 30% bovine serum, vol/vol); (ii) it is stable for 45 minutes at 100 °C; (iii) it is stable to lyophilisation; (iv) it has a negative charge and binds to an anion exchange resin;
- the step of detecting the presence of any cultured bacteria may comprise the step of identifying any cultured bacteria.
- the identification step may be a positive or negative one, e.g. it may positively identify bacteria (for example as being of a particular taxonomy such as strain, species or genus) or it may negatively identify them, confirming that they, are not of a particular taxonomy such as a particular strain, species or genus.
- the culturing and detection steps may for example be combined at least in part by the use of culture media which will affect the growth of certain bacteria.
- culture media which will affect the growth of certain bacteria.
- B. anthracis typically appears as dull white colonies of ca.4 mm diameter that are not haemolytic on blood agar plates and are not coloured on chromogenic plating media.
- B. cereus or B. thuringiensis show large haemolytic zones on blood agar and have blue-turquoise colonies on the chromogenic plating media, while growth of B. megaterium and B. subtilis is inhibited by both types of media, ⁇ -phage plaques can also be used as detailed below and are readily recognisable in the bacterial lawn as early as 4-5 hours.
- the bacterial autoinducers used in the present invention are known to display a large degree of promiscuity in the bacteria upon which they induce an effect, inducing the growth of bacterial species other than those from which they were isolated (as above, the autoinducer induces the growth of B. anthracis), the growth of all bacterial species is not induced by the same autoinducer. Therefore if a bacterial autoinducer is used which is known not to induce the growth of a given species of bacteria and bacteria are successfully cultured then it may be concluded that the bacterial spores were not those of the given species.
- the identity of a bacterium may be determined by contacting the bacterium with a binding agent specific to a target bacterium and detecting any interactions between the binding agent and the cultured bacterium.
- a binding agent may for example comprise an antibody or antigen binding fragment thereof against a target bacterium. Alternatively, it may comprise any other moiety which is specific to the target bacterium, for example a nucleotide sequence.
- the presence of a nucleotide sequence specific for a target bacterium may be determined by contacting the cultured bacterium with a specific nucleotide sequence which may hybridise to the target bacterium nucleotide sequence, or a transcription product thereof and detecting any specific hybridising reaction.
- the step of detecting the presence of any cultured bacteria may comprise the steps of: (i) contacting said cultured sample with a reagent specific to a target bacterium, the reagent being selected from the group consisting of:
- step (B) a nucleotide sequence; and (ii) detecting any antibody-antigen binding reaction, or nucleotide sequence hybridisation reaction; correlation step (e) comprising the step of correlating the results of detection step (d) with the presence or absence of said target bacterium.
- Specific rRNA hybridisation probes may be used to identify a target bacterium in a sample - these probes take advantage of the high copy numbers of rRNA present in bacterial cells, and the fact that the sample contains increased bacterial cell numbers associated with the germination and culturing of bacterial spores in the presence of autoinducer.
- antibody in its various grammatical forms is used herein to refer to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e. molecules that contain an antibody combining site or paratope. Such molecules are also referred to as "antigen binding fragments" of immunoglobulin molecules.
- Illustrative antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and those portions of an immunoglobulin molecule that contain the paratope, including those portions known in the art as Fab, Fab', F(ab')2, scFv and F(v) (Harlow, E. and Lane, D., "Using Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Press, New York, 1998).
- Also provided is a method of identifying bacterial spores in a sample according to the present invention, the step of detecting the presence of any cultured bacteria comprising the steps of:
- step (i) contacting said cultured sample with a nucleotide sequence or a complement thereof, said nucleotide sequence comprising at least one primer which is complementary to a region of a target nucleic acid which is specific to a target bacterium; and (ii) detecting any nucleotide sequence hybridisation reaction; correlation step (e) comprising the step of correlating the results of detection step (d) with the presence or absence of said target bacterium.
- the step of detecting the presence of any cultured bacteria using a nucleic acid sequence or a complement thereof comprising a primer may comprise the carrying out of an amplification reaction to amplify any of said target nucleic acid present in said sample, the detection of any nucleotide sequence hybridisation reaction comprising the detection of any amplification product of said amplification reaction.
- the amplification reaction may be a polymerase chain reaction (PCR), wherein said at least one primer may comprise a primer pair to achieve exponential amplification of the target nucleic acid (see for example GB 2367359).
- the amplification reaction may utilise a single primer to achieve linear amplification of the target nucleic acid.
- the target nucleic acid may be DNA, preferably genomic DNA. If the target nucleic acid is RNA, amplification may be achieved using reverse transcription, utilising a single primer or a primer pair (see for example Sambrook, J. et al, "Molecular Cloning. A Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor Press, New York, 1989).
- the primers for use in the PCR amplification are capable of hybridising to the target nucleic acid.
- the nucleotide sequence of the primers is designed to be complementary to the nucleotide sequence of the target nucleic acid.
- PCR amplification may be carried out by any known method of PCR (see for example “PCR (Volume 1): A practical approach", Eds. MJ. McPherson, P. Quirke and G.R. Taylor, Oxford University Press, 1991).
- PCR Volume 1: A practical approach
- other PCR methods including (but not limited to) quantitative PCR, nested PCR and fluorescence PCR may be used.
- the target nucleic acid may comprise a ribosomal RNA gene selected from the group consisting of: 5S rRNA, 16S rRNA, and 23 S rRNA.
- Primer design may be determined by comparative analysis of gene sequences from a large number of bacteria in order to determine conserved and variable regions of the gene.
- a primer for use in detecting a single genus of a gene is designed to be complementary to variable regions of a gene that are distinct between different genera but are conserved in all members of the genus of interest.
- a primer for use in detecting bacteria of a single species is designed to be complementary to variable regions of a gene that are distinct between different species but conserved in all members of the species of interest.
- a primer for use in detecting bacteria of a single strain is designed to be complementary to variable regions of that gene that are unique to the strain of interest. Primers may also be degenerate in order to hybridise to previously unidentified bacterial target nucleic acid sequences.
- the detection step may additionally comprise the step of sequencing said amplification reaction amplification product to generate sequence data
- the correlation step may comprise the step of comparing said sequence data with sequence data obtained from at least one other sample or bacterium to identify said bacteria.
- 16S rRNA is highly conserved between different species of bacteria and sequencing of 16S rRNA gene sequences has been used widely to identify bacteria using non-specific primers. Comparative analysis of sequences is widely discussed in GB 2367359.
- the public databases EMBL GenBank and the Ribosome Database project contain a large amount of sequence information which may be used for the comparative analysis of gene sequences.
- 16S rRNA gene sequence data generated for a bacterium recovered from the spore state can be compared with 16S rRNA gene sequences obtained from both identified and unidentified bacteria. A positive match will identify the bacterium in question. If no match with previously reported nucleic acid sequences (e.g. rRNA gene sequences) were observed, this indicates that it is not a target bacterium and may possibly indicate potentially novel microbes. Similarly in any identification step, be it from nucleic acid sequence or otherwise, a negative result can be useful in determining what the bacteria is not. For example, the knowledge that the bacteria is not a specific genus or species can provide useful information.
- a method for generating a database using at least one sample containing bacterial spores comprising the steps of:
- the database may contain sequence data for at least one target bacterium.
- the sequence data may be DNA, RNA or amino acid sequences.
- the database may contain at least one accession number to uniquely identify the cultured bacterium.
- the data may also e.g. comprise markers and mapping data, gene expression data, genome assembly data, 3D macromolecular structural data and taxonomic data.
- a method of generating a report of the identity of a bacterium cultured from bacterial spores in a sample comprising the steps of:
- the report may contain sequence data for at least one bacterium.
- the sequence data may be DNA, RNA or amino acid sequences.
- the report may contain at least one accession number to uniquely identify the bacterium.
- the report may also contain markers and mapping data, gene expression data, genome assembly data, 3D macromolecular structural data, and taxonomic data.
- the sample used in the present invention may be prepared from one of the group consisting of: sewage, water, ice, foodstuffs, beverages, soils, lake and sea beds, bogs, mines, deposits, bore-holes, and clinical isolates such as blood, cerebrospinal fluid, urine, tissue, sputum and pus.
- the autoinducers used in the present invention to effect resuscitation include those of WO 98/53047 although others may also be used.
- the bacterial growth autoinducers of the present invention are distinct from the N-acyl homoserine lactones and the molecule of Lyte et al. (1996, supra) (for example, the molecular weight of an autoinducer according to the present invention is approximately 1000 Da, compared to the 10,000 Da of Lyte et al) (1996, supra). It is not a peptide pheromone.
- the autoinducer of the present invention is a single compound, or else forms a family of related molecules.
- the B. anthracis strains 340, 369 and 418 used in the experiments described below were from the culture collection of the Robert Koch Institute Wernigerode, Germany. All three were isolated from environmental samples.
- B. anthracis spores were routinely produced by incubating fresh bacterial isolates on nutrient agar plates containing 1% glucose and 0.01% manganese sulphate (which have been left drying for 3-4 weeks at room temperature in the dark) for four weeks at room temperature in the dark. Spores were harvested, pelleted by centrifugation and treated with 65%o isopropanol to kill any surviving vegetative cells. Spores were again pelleted by centrifugation and washed at least 4 times with sterile distilled water, and then checked by the staining procedure of Rakette, using malachite green-eosin. Spore counts were controlled by streaking dilutions of spore suspensions in distilled water onto blood agar plates. For the experiments shown in Tables 1 and 2, the inocula contained approximately 10 3 spores.
- Spores were also cultured using the above method with partially purified autoinducer prepared from serum-SAPI cultures of Yersinia ruckeri containing 50 ⁇ M noradrenaline and similar results to those detailed below were obtained.
- spore-enrichment medium comprising:
- the material suspected of containing B. anthracis spores is suspended in approximately two volumes of B. anthracis spore enrichment medium containing Al as described above. For example, 100 g of soil would be suspended in 200 ml of the spore enrichment medium. 2. The suspension is heated at 70 °C for 30 minutes to kill vegetative cells of all bacterial species. This step ensures that only spores of B. anthracis or other spore- forming bacteria survive in the test suspension. Note that Al activity is heat resistant.
- the suspension is diluted to 5 times the volume with spore enrichment medium and incubated with shaking at 37 °C for up to 5 hours to allow even a very few B. anthracis (or other) spores to germinate and grow to e.g. 10 4 -10 5 viable cells/ml.
- the resulting culture is streaked onto commercially available medium such as blood agar medium, e.g. Anthrax-Blutagar (Heipha, Eppelheim, Germany) and onto chromogenic plating media such as Cereus-Ident-Agar (Heipha) or BCM (RTM) Bacillus cereus/Bacillus thuringiensis plating medium (Biosynth AG, Staad, Switzerland).
- blood agar medium e.g. Anthrax-Blutagar (Heipha, Eppelheim, Germany) and onto chromogenic plating media such as Cereus-Ident-Agar (Heipha) or BCM (RTM) Bacillus cereus/Bacillus thuringiensis plating medium (Biosynth AG, Staad, Switzerland).
- chromogenic plating media such as Cereus-Ident-Agar (Heipha) or BCM (RTM) Bacillus cereus/Bacillus thuringiensis plating medium (Bios
- Pinpoint colonies of Bacillus species are detectable following incubation at 37 °C for 4-5 hours.
- the presence of B. anthracis can be confirmed by spotting a species- specific ⁇ -phage onto inoculated plates in at least four different positions; a phage reaction is observable within the growth of B. anthracis after 4-5 hours, thus providing early warning of the presence of B. anthracis spores in the test sample in about 10-11 hours from the commencement of testing.
- Colonies can be further characterised by species-specific PCR-based tests, for example that detect the presence of the virulence plasmids pXO 1 and pX02.
- B. anthracis typically appears as dull white colonies that are not haemolytic on blood agar plates and are not coloured on chromogenic plating media.
- the ⁇ -phage plaques are readily recognisable in the bacterial lawn at this stage.
- B. cereus or B. thuringiensis show large haemolytic zones on blood agar and have blue-turquoise colonies on the chromogenic plating media, while growth of B. megaterium and B. subtilis is inhibited by both types of media.
- Incubation at Step 3 can be performed for a longer period of time in order to maximise recovery if a less rapid detection process is acceptable.
- steps 4-6 involve commercially available products and well-established discriminatory culture methods for Bacillus species
- steps 1-3 represent the significant new and non-obvious technical advantages of the present invention, by exploiting the enhanced speed and sensitivity of detection afforded by supplementation with bacterial autoinducer.
- the methodology may also be applicable to other spore-forming pathogenic bacterial species, particularly the spore-forming genus Clostridium.
- This genus contains a number of highly pathogenic species, including C. perfringens (the cause of gas gangrene), C. tetani (tetanus) and C. botulinum (botulism), whose spores are long lasting and widespread in soil and water.
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Abstract
The present invention is concerned with methods for the rapid detection of the presence of bacterial spores in a sample and with the identification of the bacterial spores, using bacterial growth autoinducers.
Description
Rapid Detection of Bacterial Spores
The present invention is concerned with methods for the rapid detection of the presence of bacterial spores in a sample and with the identification of the bacterial spores.
Bacillus anthracis is an aerobic, non-motile Gram-positive rod. Most members of the genus Bacillus are saprophytic, and are present in soil, water, air and vegetation. B. cereus is an important cause of food poisoning, but B. anthracis is the most virulent and best- known pathogen. Its virulence is based on two plasmids, pXOl which codes for the protective antigen, oedema factor and lethal factor, that comprise anthrax toxin, and pX02 which encodes the protective capsule.
Many species of Bacillus, including B. anthracis, form heat-resistant spores that can survive for many years in soil and ground water, and it is this property that makes B. anthracis a particular focus for the development of biological weapons. Similarly, other spore-forming bacteria include those of the genus Clostridium. This genus contains a number of highly pathogenic species, including C. perfringens (the cause of gas gangrene), C. tetani (tetanus) and C. botulinum (botulism).
Anthrax affects herbivores such as sheep, goats, cattle and horses. Bacteria are excreted in the faeces and urine of infected animals, and may form spores in the environment. Human infection may occur by contact with infected animals or contaminated animal products (hides, wool, bone meal, etc.), or by inhalation of spores.
B. anthracis spores germinate in tissues at the site of entry or in alveolar macrophages when inhaled. The bacteria multiply and produce anthrax toxin, leading to oedema.
Bacteria spread to the lymphatics and may reach the blood stream; continued bacterial multiplication and toxin production may cause generalised toxic effects, oedema and death.
Normally anthrax is rare in developed countries and when it does occur it is relatively easily diagnosed by typical culture on blood agar plates and other selective nutrient media, and it can be successfully treated by early administration of high doses of penicillin or ciprofioxacin. In the scenario in which B. anthracis spores might be deliberately and maliciously dispersed, however, there is an urgent need for rapid and sensitive techniques for environmental testing for B. anthracis spores, in order to pre-empt widespread human infection.
Currently it is a slow process to test a sample for the presence of spores, let alone to identify the bacteria which are in spore form. It is of course possible to use high quality microscopes to visualise the presence of spores under high magnification, but this requires the presence and skilled use of costly and delicate equipment. Furthermore this technique does not provide any way to identify the actual bacterium or bacteria which are in spore form. For this to be done the spores must be cultured in an attempt to induce their germination and sufficient growth of the bacterium that it may be identified. Obviously this can be costly and slow. The technique of the present invention, as detailed below, is particularly useful in detecting small numbers of bacterial spores which could simply not be detected with current techniques such as microscopy.
Signalling events between bacteria and host cells are an integral component of the dynamic and complex process of infection and disease. It has recently become clear that signalling between bacteria is also of importance to this process. Such cell-cell communication in bacteria is accomplished through the exchange of extracellular signalling molecules, typically low molecular weight compounds called autoinducers. This process, termed quorum sensing, allows bacterial populations to coordinate gene expression. This
community cooperation probably enhances the effectiveness of processes such as virulence factor expression, antibiotic resistance and biofϊlm development (Chen, X., et ah, Nature 2002, vol 415, p545-549, PMID: 11823863).
One class of autoinducers which has already been well characterised is the N-acyl homoserine lactones, which are composed of derivatives of amino acid and fatty acid molecules. This family of molecules plays a key role in the mechanisms by which Gram-negative bacteria monitor population densities, factors that are important in the virulence of a number species.
A purported bacterial autoinducer was also isolated by Lyte, M., et al. (FEMS Microbiol. Lett. 1996 Jun 1; 139(2-3): 155-9, PMID: 8674983) having a molecular weight of approximately 10,000 Da (see also Lyte, M.J., Endocrinol. 1993 Jun;137(3):343-5, PMID: 8371072; US 5629349).
As detailed in WO 98/53047, a novel family of autoinducers has been isolated, purified and characterised from various bacteria, including Escherichia coli, Hafnia alvei, and Salmonella enterica serovars, for example Enteriditis and Typhimurium. See also Freestone, P.P., et al. (FEMS Microbiol. Lett. 1999 Mar l;172(l):53-60, PMID: 10079527), and Freestone, P.P., et al. (J. Bacteriol. 2000 Nov;182(21):6091-8, PMID: 11029429).
The present inventors have now found that the family of autoinducers identified in WO 98/53047 can be useful in effecting a rapid and convenient identification of the presence of bacterial spores in a sample. This efficacy of the autoinducer in inducing growth of bacterial spores has not been previously disclosed nor has it been suggested.
In particular, although WO 98/53047 states that the autoinducer may be used to assay viable but non-culturable bacteria, no mention is made of culturing bacterial spores using the autoinducer.
Thus the present invention provides a method to do this and seeks to overcome the prior art disadvantages.
According to a first aspect of the present invention there is provided a method for culturing bacterial spores in a sample, comprising the steps of: (a) contacting said sample with a bacterial growth autoinducer, said bacterial growth autoinducer being characterised in that it has the following properties:
(i) it can be produced by a Gram-negative bacterium in response to noradrenaline in serum SAPI medium (2.77 mM glucose, 6.25 mM NH4N03, 1.84 mM KH2P04, 3.35 mM KC1, 1.01 mM
MgS04 at pH 7.5 supplemented with 30% bovine serum, vol/vol); (ii) it is stable for 45 minutes at 100 °C; (iii) it is stable to lyophilisation; (iv) it has a negative charge and binds to an anion exchange resin;
(v) it is polar and hydrophilic, is soluble in water, and substantially insoluble in methanol, propanol, butanol, n-hexane, CHC13 and DMSO; and (vi) it has a molecular weight of approximately 1000 Daltons; and
(b) culturing said sample
In order to culture only the bacterial spores contained in said sample, method step (a) may be preceded by the step of treating said sample to inactivate micro-organisms (for example
to kill them) contained therein other than bacterial spores. The treatment may be to inactivate bacteria other than those in spore form. The inactivation treatment may for example kill bacteria such as Bacillus species not in spore form and other micro-organisms including fungi.
The inactivation treatment may be anything which does not inactivate bacterial spores but which does inactivate other chosen micro-organisms. It may for example be a heat- treatment which inactivates bacteria not in spore form - a sample can for example be heated to 70 °C for a period of at least 5 minutes, for example at least 10, 15, 20, 25 or 30 minutes. Alternatively, an alcohol such as iso-propanol can be used, as can disinfectants and other techniques as appropriate such as sonication.
The bacterial growth autoinducer may further be characterised in having at least one of the following characteristics: (a) it is stable in prolonged storage in a dried state and/or in solution; and
(b) it is stable when autoclaved.
The bacterial growth autoinducer may further be characterised in having at least one of the following characteristics: (a) it can be produced by bacteria grown in Luria broth, tryptone soya broth, M9 minimal medium and Davis-Mingioli minimal medium as well as by the same bacteria grown in serum SAPI medium;
(b) it has a reddish-pink colour;
(c) it contains serine; (d) its synthesis is not stimulated by conditions of Fe starvation;
(e) it is synthesised in conditions of excess Fe;
(f) its entry into bacteria occurs via a TonB dependent receptor; and
(g) it is resistant to degradation by ribonuclease, deoxyribonuclease, trypsin, pepsin, N8 protease, proteinase K, acid phosphatases, alkaline phosphatase and phosphodiesterase.
Examples of bacterial growth autoinducers which can be used in the methods of the present invention include those isolated from Escherichia coli, Yersinia ruckeri, Hafnia alvei, and Salmonella enterica serovars Enteriditis and Typhimurium. As is known from the prior art, the autoinducers are typically not species-specific, instead an autoinducer from one species being promiscuous and often being able to induce the growth of bacteria from a range of other species. As is shown in the experiments below, the germination and culturing of B. anthracis spores is induced by the autoinducer. Similarly, the present invention is not limited to the use of autoinducers from the above list of species alone, but may include other species of the family Enter obacteriaceae.
Culturing may be performed using conventional culture media in addition to the bacterial autoinducer.
According to a second aspect of the present invention there is also provided a method for the detection of bacterial spores in a sample, comprising the steps of: (a) treating said sample to inactivate bacteria contained in it which are not in spore-form; (b) contacting said treated sample with a bacterial growth autoinducer, said bacterial growth autoinducer being characterised in that it has the following properties: (i) it can be produced by a Gram-negative bacterium in response to noradrenaline in serum SAPI medium (2.77 mM glucose, 6.25 mMΝH4Ν035 1.84 mM KH2P04, 3.35 mM KCl, 1.01 mM
MgS04 at pH 7.5 supplemented with 30% bovine serum, vol/vol); (ii) it is stable for 45 minutes at 100 °C; (iii) it is stable to lyophilisation; (iv) it has a negative charge and binds to an anion exchange resin;
(v) it is polar and hydrophilic, is soluble in water, and substantially insoluble in methanol, propanol, butanol, n-hexane, CHC13 and
DMSO; and (vi) it has a molecular weight of approximately 1000 Daltons; (c) culturing said sample
(d) detecting the presence of any cultured bacteria; and
(e) correlating the results of detection step (d) with the presence of bacterial spores in said sample.
The step of detecting the presence of any cultured bacteria may comprise the step of identifying any cultured bacteria. The identification step may be a positive or negative one, e.g. it may positively identify bacteria (for example as being of a particular taxonomy such as strain, species or genus) or it may negatively identify them, confirming that they, are not of a particular taxonomy such as a particular strain, species or genus.
The culturing and detection steps may for example be combined at least in part by the use of culture media which will affect the growth of certain bacteria. For example after incubation to a total of 18-20 hours, B. anthracis typically appears as dull white colonies of ca.4 mm diameter that are not haemolytic on blood agar plates and are not coloured on chromogenic plating media. In contrast, B. cereus or B. thuringiensis show large haemolytic zones on blood agar and have blue-turquoise colonies on the chromogenic plating media, while growth of B. megaterium and B. subtilis is inhibited by both types of
media, γ-phage plaques can also be used as detailed below and are readily recognisable in the bacterial lawn as early as 4-5 hours.
Although the bacterial autoinducers used in the present invention are known to display a large degree of promiscuity in the bacteria upon which they induce an effect, inducing the growth of bacterial species other than those from which they were isolated (as above, the autoinducer induces the growth of B. anthracis), the growth of all bacterial species is not induced by the same autoinducer. Therefore if a bacterial autoinducer is used which is known not to induce the growth of a given species of bacteria and bacteria are successfully cultured then it may be concluded that the bacterial spores were not those of the given species.
After culturing, the identity of a bacterium may be determined by contacting the bacterium with a binding agent specific to a target bacterium and detecting any interactions between the binding agent and the cultured bacterium. A binding agent may for example comprise an antibody or antigen binding fragment thereof against a target bacterium. Alternatively, it may comprise any other moiety which is specific to the target bacterium, for example a nucleotide sequence. The presence of a nucleotide sequence specific for a target bacterium may be determined by contacting the cultured bacterium with a specific nucleotide sequence which may hybridise to the target bacterium nucleotide sequence, or a transcription product thereof and detecting any specific hybridising reaction.
Thus the step of detecting the presence of any cultured bacteria may comprise the steps of: (i) contacting said cultured sample with a reagent specific to a target bacterium, the reagent being selected from the group consisting of:
(A) an antibody or antigen binding fragment thereof; and
(B) a nucleotide sequence; and
(ii) detecting any antibody-antigen binding reaction, or nucleotide sequence hybridisation reaction; correlation step (e) comprising the step of correlating the results of detection step (d) with the presence or absence of said target bacterium.
Specific rRNA hybridisation probes may be used to identify a target bacterium in a sample - these probes take advantage of the high copy numbers of rRNA present in bacterial cells, and the fact that the sample contains increased bacterial cell numbers associated with the germination and culturing of bacterial spores in the presence of autoinducer.
The term "antibody" in its various grammatical forms is used herein to refer to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e. molecules that contain an antibody combining site or paratope. Such molecules are also referred to as "antigen binding fragments" of immunoglobulin molecules.
Illustrative antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and those portions of an immunoglobulin molecule that contain the paratope, including those portions known in the art as Fab, Fab', F(ab')2, scFv and F(v) (Harlow, E. and Lane, D., "Using Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Press, New York, 1998).
Also provided is a method of identifying bacterial spores in a sample according to the present invention, the step of detecting the presence of any cultured bacteria comprising the steps of:
(i) contacting said cultured sample with a nucleotide sequence or a complement thereof, said nucleotide sequence comprising at least one
primer which is complementary to a region of a target nucleic acid which is specific to a target bacterium; and (ii) detecting any nucleotide sequence hybridisation reaction; correlation step (e) comprising the step of correlating the results of detection step (d) with the presence or absence of said target bacterium.
The step of detecting the presence of any cultured bacteria using a nucleic acid sequence or a complement thereof comprising a primer may comprise the carrying out of an amplification reaction to amplify any of said target nucleic acid present in said sample, the detection of any nucleotide sequence hybridisation reaction comprising the detection of any amplification product of said amplification reaction.
The amplification reaction may be a polymerase chain reaction (PCR), wherein said at least one primer may comprise a primer pair to achieve exponential amplification of the target nucleic acid (see for example GB 2367359). Alternatively, the amplification reaction may utilise a single primer to achieve linear amplification of the target nucleic acid. The target nucleic acid may be DNA, preferably genomic DNA. If the target nucleic acid is RNA, amplification may be achieved using reverse transcription, utilising a single primer or a primer pair (see for example Sambrook, J. et al, "Molecular Cloning. A Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor Press, New York, 1989). The primers for use in the PCR amplification are capable of hybridising to the target nucleic acid. The nucleotide sequence of the primers is designed to be complementary to the nucleotide sequence of the target nucleic acid. PCR amplification may be carried out by any known method of PCR (see for example "PCR (Volume 1): A practical approach", Eds. MJ. McPherson, P. Quirke and G.R. Taylor, Oxford University Press, 1991). In addition to standard PCR reactions, other PCR methods including (but not limited to) quantitative PCR, nested PCR and fluorescence PCR may be used.
The target nucleic acid may comprise a ribosomal RNA gene selected from the group consisting of: 5S rRNA, 16S rRNA, and 23 S rRNA. Primer design may be determined by comparative analysis of gene sequences from a large number of bacteria in order to determine conserved and variable regions of the gene. A primer for use in detecting a single genus of a gene is designed to be complementary to variable regions of a gene that are distinct between different genera but are conserved in all members of the genus of interest. A primer for use in detecting bacteria of a single species is designed to be complementary to variable regions of a gene that are distinct between different species but conserved in all members of the species of interest. A primer for use in detecting bacteria of a single strain is designed to be complementary to variable regions of that gene that are unique to the strain of interest. Primers may also be degenerate in order to hybridise to previously unidentified bacterial target nucleic acid sequences.
The detection step may additionally comprise the step of sequencing said amplification reaction amplification product to generate sequence data, and the correlation step may comprise the step of comparing said sequence data with sequence data obtained from at least one other sample or bacterium to identify said bacteria. 16S rRNA is highly conserved between different species of bacteria and sequencing of 16S rRNA gene sequences has been used widely to identify bacteria using non-specific primers. Comparative analysis of sequences is widely discussed in GB 2367359. The public databases EMBL GenBank and the Ribosome Database project contain a large amount of sequence information which may be used for the comparative analysis of gene sequences. For example, 16S rRNA gene sequence data generated for a bacterium recovered from the spore state can be compared with 16S rRNA gene sequences obtained from both identified and unidentified bacteria. A positive match will identify the bacterium in question. If no match with previously reported nucleic acid sequences (e.g. rRNA gene sequences) were observed, this indicates that it is not a target bacterium and may possibly indicate potentially novel microbes. Similarly in any identification step, be it from nucleic acid
sequence or otherwise, a negative result can be useful in determining what the bacteria is not. For example, the knowledge that the bacteria is not a specific genus or species can provide useful information.
According to a third aspect of the present invention there is provided a method for generating a database using at least one sample containing bacterial spores, comprising the steps of:
(a) contacting said sample with a bacterial growth autoinducer, said bacterial growth autoinducer being characterised in that it has the following properties:
(i) it can be produced by a Gram-negative bacterium in response to noradrenaline in serum SAPI medium (2.77 mM glucose,
6.25 mMNH4N03, 1.84 mM KH2P04, 3.35 mM KC1, 1.01 mM
MgS04 at pH 7.5 supplemented with 30% bovine serum, vol/vol);
(ii) it is stable for 45 minutes at 100 °C; (iii) it is stable to lyophilisation;
(iv) it has a negative charge and binds to an anion exchange resin;
(v) it is polar and hydrophilic, is soluble in water, and substantially insoluble in methanol, propanol, butanol, n-hexane, CHC13 and
DMSO; and (vi) it has a molecular weight of approximately 1000 Daltons; and
(b) culturing said sample;
(c) contacting said cultured sample with a nucleotide sequence or a complement thereof, the nucleotide sequence comprising at least one primer which is complementary to a region of a target nucleic acid which is specific to a target bacterium;
(d) carrying out an amplification reaction to amplify any of said target nucleic acid present in said cultured sample;
(e) detecting any amplification product of said amplification reaction by sequencing said amplification reaction amplification product to generate sequence data; and
(f) compiling said sequence data onto said database.
The database may contain sequence data for at least one target bacterium. The sequence data may be DNA, RNA or amino acid sequences. Alternatively the database may contain at least one accession number to uniquely identify the cultured bacterium. The data may also e.g. comprise markers and mapping data, gene expression data, genome assembly data, 3D macromolecular structural data and taxonomic data.
According to a fourth aspect of the present invention there is provided a method of generating a report of the identity of a bacterium cultured from bacterial spores in a sample comprising the steps of:
(a) identifying bacteria cultured from bacterial spores in a sample according to the method of the present invention; and
(b) generating a report of the identity of said cultured bacterium incorporating the results of said method to identify said cultured bacterium.
The report may contain sequence data for at least one bacterium. The sequence data may be DNA, RNA or amino acid sequences. Alternatively the report may contain at least one accession number to uniquely identify the bacterium. The report may also contain markers and mapping data, gene expression data, genome assembly data, 3D macromolecular structural data, and taxonomic data.
The sample used in the present invention may be prepared from one of the group consisting of: sewage, water, ice, foodstuffs, beverages, soils, lake and sea beds, bogs, mines, deposits, bore-holes, and clinical isolates such as blood, cerebrospinal fluid, urine, tissue, sputum and pus.
The autoinducers used in the present invention to effect resuscitation include those of WO 98/53047 although others may also be used.
The bacterial growth autoinducers of the present invention are distinct from the N-acyl homoserine lactones and the molecule of Lyte et al. (1996, supra) (for example, the molecular weight of an autoinducer according to the present invention is approximately 1000 Da, compared to the 10,000 Da of Lyte et al) (1996, supra). It is not a peptide pheromone. As detailed in WO 98/53047, experiments have shown that the autoinducer of the present invention is a single compound, or else forms a family of related molecules.
As is demonstrated by the experiments detailed below, the treatment of a sample containing bacterial spores with such an autoinducer results in the germination and culturing of the bacteria, thereby enabling their detection using standard techniques such as plating on solid microbiological media. This use of the bacterial growth autoinducer to culture bacterial spores has not been previously suggested."
It provides advantages over existing media in that it induces the germination and growth of bacterial spores, including Bacillus spores, and the rate of growth of the germinated bacteria may be greater than that achieved using other methods.
Hence the present invention is not only an alternative to existing methods but also provides substantial advantages over them.
The contents of each of the references discussed herein, including the references cited therein, are herein incorporated by reference in their entirety. Where "PMID" reference numbers are given for publications, these are the PubMed identification numbers allocated to them by the US National Library of Medicine, from which full bibliographic information and abstract for each publication is available at www.ncbi.nlm.nih.gov.
The invention will be further apparent from the following description, which shows, by way of example only, germination and culturing of bacterial spores from various sources.
EXPERJMENTS
The following experiments were undertaken to show the induction of germination and subsequent growth of bacterial spores that can be achieved using the bacterial autoinducers of the present invention, and the rapid and effective detection and identification of bacterial spores that this allows.
Materials and Methods
Bacterial strains
The B. anthracis strains 340, 369 and 418 used in the experiments described below were from the culture collection of the Robert Koch Institute Wernigerode, Germany. All three were isolated from environmental samples.
Preparation of spores
B. anthracis spores were routinely produced by incubating fresh bacterial isolates on nutrient agar plates containing 1% glucose and 0.01% manganese sulphate (which have been left drying for 3-4 weeks at room temperature in the dark) for four weeks at room temperature in the dark. Spores were harvested, pelleted by centrifugation and treated with 65%o isopropanol to kill any surviving vegetative cells. Spores were again pelleted by centrifugation and washed at least 4 times with sterile distilled water, and then checked by the staining procedure of Rakette, using malachite green-eosin. Spore counts were controlled by streaking dilutions of spore suspensions in distilled water onto blood agar plates. For the experiments shown in Tables 1 and 2, the inocula contained approximately 103 spores.
Culture conditions:
Approximately 103 spores, prepared as described, were inoculated into 5 ml lots of buffered peptone water (BPW):
(i) containing no supplements;
(ii) supplemented with 1% (vol/vol) partially purified autoinducer (Al) prepared from serum-SAPI cultures of Escherichia coli containing 50 μM noradrenaline (Nor), as described previously in WO 98/53047; and (iii) supplemented with 50 μM Nor.
Cultures were incubated at 37 °C by shaking (150 rpm, longitudinal) for the periods of time shown in the Tables (below), following which culture turbidity (OD620) was determined spectrophotometrically directly in the culture tubes themselves. All experimental values shown below are based on duplicate samples.
Spores were also cultured using the above method with partially purified autoinducer prepared from serum-SAPI cultures of Yersinia ruckeri containing 50 μM noradrenaline and similar results to those detailed below were obtained.
Results Tables 1 and 2 show data from two independent experiments.
In the experiments shown in Tables 1 and 2, supplementation of BPW with partially purified bacterial autoinducer significantly enhanced the germination and growth of spores compared to unsupplemented BPW at all time-points tested. The possibility that the observed results are due to a carry-over of residual noradrenaline from the serum-SAPI medium used to prepare autoinducer is ruled out by the observation in Table 2 that supplementation with as much as 50 μM noradrenaline did not enhance spore germination and growth to the same extent as 1% bacterial autoinducer. Overall these data indicate that
supplementation of enrichment medium with bacterial autoinducer improves speed and sensitivity of detection of B. anthracis spores.
To maximise the sensitivity of detection of low numbers of Bacillus anthracis spores in clinical or environmental samples, a "spore-enrichment medium" was developed comprising:
8.0 g/1 of nutrient broth (Beckton Dickinson)
5.0 g/1 tryptone
5.0 g/l NaCl 4.0 g/l K2HPO4
2.0 g/1 glucose
0.2%) (vol/vol) partially purified enterobacterial autoinducer of growth (Al), prepared as described in WO 98/53047
Incubation by shaking at 37 °C of a small number of spores in this medium resulted in consistent germination and multiplication to 104- 105 viable cells/ml within 4-5 hours, sufficient for further cultural and molecular methods as described herein. In the absence of Al, growth occurs significantly more slowly.
Discussion
On the basis of these data the following method may be used for the rapid cultural diagnosis of B. anthracis in clinical and environmental samples:
1. The material suspected of containing B. anthracis spores is suspended in approximately two volumes of B. anthracis spore enrichment medium containing Al as described above. For example, 100 g of soil would be suspended in 200 ml of the spore enrichment medium.
2. The suspension is heated at 70 °C for 30 minutes to kill vegetative cells of all bacterial species. This step ensures that only spores of B. anthracis or other spore- forming bacteria survive in the test suspension. Note that Al activity is heat resistant.
3. The suspension is diluted to 5 times the volume with spore enrichment medium and incubated with shaking at 37 °C for up to 5 hours to allow even a very few B. anthracis (or other) spores to germinate and grow to e.g. 104-105 viable cells/ml.
4. The resulting culture is streaked onto commercially available medium such as blood agar medium, e.g. Anthrax-Blutagar (Heipha, Eppelheim, Germany) and onto chromogenic plating media such as Cereus-Ident-Agar (Heipha) or BCM (RTM) Bacillus cereus/Bacillus thuringiensis plating medium (Biosynth AG, Staad, Switzerland).
5. Pinpoint colonies of Bacillus species are detectable following incubation at 37 °C for 4-5 hours. The presence of B. anthracis can be confirmed by spotting a species- specific γ-phage onto inoculated plates in at least four different positions; a phage reaction is observable within the growth of B. anthracis after 4-5 hours, thus providing early warning of the presence of B. anthracis spores in the test sample in about 10-11 hours from the commencement of testing. Colonies can be further characterised by species-specific PCR-based tests, for example that detect the presence of the virulence plasmids pXO 1 and pX02.
6. After further incubation to a total of 18-20 hours, B. anthracis typically appears as dull white colonies that are not haemolytic on blood agar plates and are not coloured on chromogenic plating media. The γ-phage plaques are readily recognisable in the bacterial lawn at this stage. In contrast, B. cereus or B. thuringiensis show large haemolytic zones on blood agar and have blue-turquoise colonies on the chromogenic
plating media, while growth of B. megaterium and B. subtilis is inhibited by both types of media.
Incubation at Step 3 can be performed for a longer period of time in order to maximise recovery if a less rapid detection process is acceptable.
Note that, while steps 4-6 involve commercially available products and well-established discriminatory culture methods for Bacillus species, steps 1-3 represent the significant new and non-obvious technical advantages of the present invention, by exploiting the enhanced speed and sensitivity of detection afforded by supplementation with bacterial autoinducer.
The methodology may also be applicable to other spore-forming pathogenic bacterial species, particularly the spore-forming genus Clostridium. This genus contains a number of highly pathogenic species, including C. perfringens (the cause of gas gangrene), C. tetani (tetanus) and C. botulinum (botulism), whose spores are long lasting and widespread in soil and water.
Table 1: Effect of Al on germination and growth of B. anthracis spores
Strain no. Incubation BPW BPW + AI time (h) (ODfi7n) (ODfi?n)
369 4.0 0.109 0.187
6.5 0.251 0.604
340 4.0 0.103 0.171
6.5 0.341 0.670
418 4.0 0.069 0.276
6.5 0.461 0.847
Table 2: Effect of Al and Nor on germination and growth of B. anthr
Strain no. Incubation BPW BPW + AI BPW + Nor time (h) (ODfi20) (OD (OD67n)
369 4.25 0.132 0.392 0.190 6.75 0.777 0.985 0.908
418 4.25 0.075 0.167 0.118
Claims
1. A method for culturing bacterial spores in a sample, comprising the steps of:
(a) contacting said sample with a bacterial growth autoinducer, said bacterial growth autoinducer being characterised in that it has the following properties:
(i) it can be produced by a Gram-negative bacterium in response to noradrenaline in serum SAPI medium (2.77 mM glucose,
6.25 mMNH4N03, 1.84
3.35 mM KCl, 1.01 mM MgS04 at pH 7.5 supplemented with 30% bovine serum, vol/vol); (ii) it is stable for 45 minutes at 100 °C; (iii) it is stable to lyophilisation;
(iv) it has a negative charge and binds to an anion exchange resin; (v) it is polar and hydrophilic, is soluble in water, and substantially insoluble in methanol, propanol, butanol, n-hexane, CHC13 and
DMSO; and (vi) it has a molecular weight of approximately 1000 Daltons; and
(b) culturing said sample
2. A method according to claim 1 , step (a) being preceded by the step of treating said sample to inactivate micro-organisms other than bacterial spores contained therein.
3. A method according to claim 2, said treatment being to inactivate bacteria other than those in spore form.
4. A method according to either of claims 2 or 3, said treatment being a heat- treatment which inactivates bacteria not in spore form.
5. A method according to claim 4, said sample being heated to 70 ° C for at least a period selected from the group consisting of: 5, 10, 15, 20, 25 and 30 minutes.
6. A method according to either of claims 2 or 3, said treatment being selected from the group consisting of treatment of said sample with: an alcohol, a disinfectant, and sonication.
7. A method according to any of the preceding claims, the bacterial growth autoinducer being further characterised in having at least one of the following characteristics:
(a) it is stable in prolonged storage in a dried state and/or in solution; and
(b) it is stable when autoclaved.
8. A method according to any of the preceding claims, the bacterial growth autoinducer being further characterised in having at least one of the following characteristics:
(a) it can be produced by bacteria grown in Luria broth, tryptone soya broth, M9 minimal medium and Davis-Mingioli minimal medium as well as by the same bacteria grown in serum SAPI medium; (b) it has a reddish-pink colour;
(c) it contains serine;
(d) its synthesis is not stimulated by conditions of Fe starvation;
(e) it is synthesised in conditions of excess Fe;
(f) its entry into bacteria occurs via a ToriB dependent receptor; and (g) it is resistant to degradation by ribonuclease, deoxyribonuclease, trypsin, pepsin, V8 protease, proteinase K, acidphosphatases, alkaline phosphatase and phosphodiesterase.
9. A method according to any of the preceding claims, the bacterial growth autoinducer being from a bacterium selected from the group consisting of: Escherichia coli, Yersinia ruckeri, Hafiiia alvei, Salmonella enterica serovar Enteriditis, and Salmonella enterica serovar Typhimurium.
10. A method according to any of the preceding claims, culturing being performed using conventional culture media in addition to said bacterial autoinducer.
11. A method for the detection of bacterial spores in a sample, comprising the steps of:
(a) treating said sample to inactivate bacteria contained in it which are not in spore-form;
(b) contacting said treated sample with a bacterial growth autoinducer, said bacterial growth autoinducer being characterised in that it has the following properties:
(i) it can be produced by a Gram-negative bacterium in response to noradrenaline in serum SAPI medium (2.77 mM glucose,
6.25 mMNH4N03, 1.84
3.35 mM KC1, 1.01 mM
MgS04 at pH 7.5 supplemented with 30% bovine serum, vol/vol);
(ii) it is stable for 45 minutes at 100 °C; (iii) it is stable to lyophilisation;
(iv) it has a negative charge and binds to an anion exchange resin;
(v) it is polar and hydrophilic, is soluble in water, and substantially insoluble in methanol, propanol, butanol, n-hexane, CHC13 and
DMSO; and (vi) it has a molecular weight of approximately 1000 Daltons;
(c) culturing said sample
(d) detecting the presence of any cultured bacteria; and
(e) correlating the results of detection step (d) with the presence of bacterial spores in said sample.
12. A method according to claim 11 , step (d) comprising the step of identifying any cultured bacteria.
13. A method according to claim 11 , step (d) comprising the steps of:
(i) contacting said cultured sample with a reagent specific to a target bacterium, the reagent being selected from the group consisting of:
(A) an antibody or antigen binding fragment thereof; and
(B) a nucleotide sequence; and
(ii) detecting any antibody-antigen binding reaction, or nucleotide sequence hybridisation reaction; correlation step (e) comprising the step of correlating the results of detection step (d) with the presence or absence of said target bacterium.
14. A method according to claim 13, the reagent specific to a target bacterium being an rRNA hybridisation probe.
15. A method according to claim 11, the step of detecting the presence of any cultured bacteria comprising the steps of:
(i) contacting said cultured sample with a nucleotide sequence or a complement thereof, said nucleotide sequence comprising at least one primer which is complementary to a region of a target nucleic acid which is specific to a target bacterium; and (ii) detecting any nucleotide sequence hybridisation reaction;
correlation step (e) comprising the step of correlating the results of detection step (d) with the presence or absence of said target bacterium.
16. A method according to claim 15, step (i) comprising the carrying out of an amplification reaction to amplify any of said target nucleic acid present in said sample, and step (ii) comprising the detection of any amplification product of said amplification reaction.
17. A method according to claim 16, said amplification reaction being a polymerase chain reaction (PCR).
18. A method according to claim 16, said amplification reaction utilising a single primer to achieve linear amplification of said target nucleic acid.
19. A method according to any of claims 16-18, said target nucleic acid comprising a ribosomal RNA gene selected from the group consisting of: 5S rRNA, 16S rRNA, and 23 S rRNA.
20. A method of generating a report of the identity of a bacterium cultured from bacterial spores in a sample comprising the steps of:
(a) identifying bacteria cultured from bacterial spores in a sample according to the method of any of claims 12-19; and
(b) generating a report of the identity of said cultured bacterium incorporating the results of said method to identify said cultured bacterium.
21. A method for generating a database using at least one sample containing bacterial spores, comprising the steps of:
(a) contacting said sample with a bacterial growth autoinducer, said bacterial growth autoinducer being characterised in that it has the following properties:
(i) it can be produced by a Gram-negative bacterium in response to noradrenaline in serum SAPI medium (2.77 mM glucose,
6.25 mM NH4N03, 1.84 mM KH-jPO* 3.35 mM KC1, 1.01 mM MgS04 at pH 7.5 supplemented with 30% bovine serum, vol/vol); (ii) it is stable for 45 minutes at 100 °C; (iii) it is stable to lyophilisation;
(iv) it has a negative charge and binds to an anion exchange resin;
(v) it is polar and hydrophilic, is soluble in water, and substantially insoluble in methanol, propanol, butanol, n-hexane, CHC13 and
DMSO; and (vi) it has a molecular weight of approximately 1000 Daltons; and
(b) culturing said sample
(c) contacting said cultured sample with a nucleotide sequence or a complement thereof, the nucleotide sequence comprising at least one primer which is complementary to a region of a target nucleic acid which is specific to a target bacterium;
(d) carrying out an amplification reaction to amplify any of said target nucleic acid present in said cultured sample;
(e) detecting any amplification product of said amplification reaction by sequencing said amplification reaction amplification product to generate sequence data; and
(f) compiling said sequence data onto said database.
22. A method according to claim 21 , said database containing sequence data for at least one target bacterium.
23. A method according to claim 22, said sequence data being selected from the group consisting of: DNA, RNA and amino acid sequences.
24. A method according to claim 21, said database containing at least one accession number to uniquely identify said cultured bacterium.
25. A method according to any of claims 21-24, said data compiled onto said database additionally comprising data concerning at least one of the group consisting of: markers and mapping data, gene expression data, genome assembly data, 3D macromolecular structural data and taxonomic data.
26. A method according to any of the preceding claims, said sample being prepared from one of the group consisting of: sewage, water, ice, foodstuffs, beverages, soils, lake and sea beds, bogs, mines, deposits, bore-holes, and clinical isolates.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU2003283572A AU2003283572A1 (en) | 2002-11-13 | 2003-11-13 | Rapid detection of bacterial spores |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GBGB0226399.4A GB0226399D0 (en) | 2002-11-13 | 2002-11-13 | Detection of bacterial spores |
| GB0226399.4 | 2002-11-13 |
Publications (1)
| Publication Number | Publication Date |
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| WO2004044187A1 true WO2004044187A1 (en) | 2004-05-27 |
Family
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|---|---|---|---|
| PCT/GB2003/004923 WO2004044187A1 (en) | 2002-11-13 | 2003-11-13 | Rapid detection of bacterial spores |
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|---|---|
| AU (1) | AU2003283572A1 (en) |
| GB (1) | GB0226399D0 (en) |
| WO (1) | WO2004044187A1 (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998053047A1 (en) * | 1997-05-22 | 1998-11-26 | University Of Leicester | E. coli, salmonella or hafnia autoinducers |
| WO2000032747A2 (en) * | 1998-12-01 | 2000-06-08 | The University Of Leicester | Campylobacter autoinducer |
-
2002
- 2002-11-13 GB GBGB0226399.4A patent/GB0226399D0/en not_active Ceased
-
2003
- 2003-11-13 WO PCT/GB2003/004923 patent/WO2004044187A1/en not_active Application Discontinuation
- 2003-11-13 AU AU2003283572A patent/AU2003283572A1/en not_active Abandoned
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998053047A1 (en) * | 1997-05-22 | 1998-11-26 | University Of Leicester | E. coli, salmonella or hafnia autoinducers |
| WO2000032747A2 (en) * | 1998-12-01 | 2000-06-08 | The University Of Leicester | Campylobacter autoinducer |
Non-Patent Citations (2)
| Title |
|---|
| FREESTONE P P E ET AL: "STIMULATION OF BACTERIAL GROWTH BY HEAT-STABLE, NOREPINEPHRINE-INDUCED AUTOINDUCERS", FEMS MICROBIOLOGY LETTERS, AMSTERDAM, NL, vol. 172, no. 1, 1999, pages 53 - 60, XP000886983, ISSN: 0378-1097 * |
| LYTE M ET AL: "Production of an autoinducer of growth by norepinephrine cultured Escherichia coli O157:H7", FEMS MICROBIOLOGY LETTERS, AMSTERDAM, NL, vol. 139, no. 2/3, 1 June 1996 (1996-06-01), pages 155 - 159, XP002078986, ISSN: 0378-1097 * |
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| GB0226399D0 (en) | 2002-12-18 |
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