GB2429283A - Preservative efficacy testing system - Google Patents
Preservative efficacy testing system Download PDFInfo
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- GB2429283A GB2429283A GB0615270A GB0615270A GB2429283A GB 2429283 A GB2429283 A GB 2429283A GB 0615270 A GB0615270 A GB 0615270A GB 0615270 A GB0615270 A GB 0615270A GB 2429283 A GB2429283 A GB 2429283A
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- preservative efficacy
- efficacy testing
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- antimicrobial preservative
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- 238000012360 testing method Methods 0.000 title claims abstract description 42
- 239000003755 preservative agent Substances 0.000 title claims abstract description 28
- 230000002335 preservative effect Effects 0.000 title claims abstract description 28
- 244000005700 microbiome Species 0.000 claims abstract description 34
- 230000000845 anti-microbial effect Effects 0.000 claims abstract description 18
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 15
- 230000001105 regulatory effect Effects 0.000 claims abstract description 14
- 230000000813 microbial effect Effects 0.000 claims abstract description 6
- 241000222122 Candida albicans Species 0.000 claims abstract description 5
- 241000588915 Klebsiella aerogenes Species 0.000 claims abstract description 4
- 241000589517 Pseudomonas aeruginosa Species 0.000 claims abstract description 4
- 229940095731 candida albicans Drugs 0.000 claims abstract description 4
- 229940092559 enterobacter aerogenes Drugs 0.000 claims abstract description 4
- 230000035899 viability Effects 0.000 claims abstract description 4
- 241000228245 Aspergillus niger Species 0.000 claims abstract description 3
- 241000588724 Escherichia coli Species 0.000 claims abstract description 3
- 238000000034 method Methods 0.000 claims description 11
- 108700008625 Reporter Genes Proteins 0.000 claims description 6
- PCTMTFRHKVHKIS-BMFZQQSSSA-N (1s,3r,4e,6e,8e,10e,12e,14e,16e,18s,19r,20r,21s,25r,27r,30r,31r,33s,35r,37s,38r)-3-[(2r,3s,4s,5s,6r)-4-amino-3,5-dihydroxy-6-methyloxan-2-yl]oxy-19,25,27,30,31,33,35,37-octahydroxy-18,20,21-trimethyl-23-oxo-22,39-dioxabicyclo[33.3.1]nonatriaconta-4,6,8,10 Chemical compound C1C=C2C[C@@H](OS(O)(=O)=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2.O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C=C/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 PCTMTFRHKVHKIS-BMFZQQSSSA-N 0.000 claims description 3
- 239000004475 Arginine Substances 0.000 claims description 3
- 108010049152 Cold Shock Proteins and Peptides Proteins 0.000 claims description 3
- 108090001030 Lipoproteins Proteins 0.000 claims description 3
- 102000004895 Lipoproteins Human genes 0.000 claims description 3
- 102000002278 Ribosomal Proteins Human genes 0.000 claims description 3
- 108010000605 Ribosomal Proteins Proteins 0.000 claims description 3
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 3
- 239000012528 membrane Substances 0.000 claims description 3
- 102000004169 proteins and genes Human genes 0.000 claims description 3
- 238000004020 luminiscence type Methods 0.000 claims description 2
- 241000191967 Staphylococcus aureus Species 0.000 abstract description 3
- 241000235033 Zygosaccharomyces rouxii Species 0.000 abstract description 2
- 239000013612 plasmid Substances 0.000 description 9
- 241000894006 Bacteria Species 0.000 description 8
- 238000009472 formulation Methods 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- 210000004027 cell Anatomy 0.000 description 6
- 239000002537 cosmetic Substances 0.000 description 6
- 230000005526 G1 to G0 transition Effects 0.000 description 4
- 238000011161 development Methods 0.000 description 3
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- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 238000005415 bioluminescence Methods 0.000 description 2
- 230000029918 bioluminescence Effects 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000012395 formulation development Methods 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 241000228212 Aspergillus Species 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- 241000588914 Enterobacter Species 0.000 description 1
- 241000588722 Escherichia Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
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- 241000589516 Pseudomonas Species 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
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- 230000001580 bacterial effect Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 230000001332 colony forming effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000004624 confocal microscopy Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
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- 230000007613 environmental effect Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 238000013537 high throughput screening Methods 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000012009 microbiological test Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 231100000041 toxicology testing Toxicity 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000002351 wastewater Substances 0.000 description 1
Classifications
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- 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
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/18—Testing for antimicrobial activity of a material
-
- 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
- C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
-
- 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
- C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
-
- 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
-
- 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
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/6825—Nucleic acid detection involving sensors
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Engineering & Computer Science (AREA)
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- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Analytical Chemistry (AREA)
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- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Toxicology (AREA)
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- Urology & Nephrology (AREA)
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- Tropical Medicine & Parasitology (AREA)
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Abstract
A system for antimicrobial preservative efficacy testing (PET/AET) is provided, comprising a biosensor incorporating at least one microbial strain selected from the group of micro-organisms comprising a regulatory recognised key microorganism for preservative efficacy testing. The micro-organism is engineered to have a constitutive promoter linked to a lux or luc gene cassette to produce a specific detectable signal reporting the viability of the micro-organism. The micro-organism is preferably selected from Eschericia coli, Pseudomonas aeruginosa, Enterobacter aerogenes, Candida albicans, Staphylococcus aureus, Aspergillus niger and Zygosaccharomyces rouxii.
Description
Preservative Efficacy Testing
Field of the Invention
The present invention relates to a system and method for antimicrobial preservative efficacy testing (AET or PET) for the pharmaceutical and cosmetic/ toiletry industries.
Background to the Invention
Preservative efficacy testing for antimicrobial preservative activity is a regulatory requirement for pharmaceuticals and cosmetics products in most countries to support the required or claimed shelf life for the products, whether antimicrobial preservatives are incorporated as additives in such products or those products intrinsically have anti- microbial properties.
Preservative efficacy testing is normally based on conventional microbiology cell : culturing techniques in which a sample of the pharmaceutical or cosmetic product is S..
inoculated with a microbial suspension of a population of a regulatory recognised key micro-organism, normally being one of the bacteria Escherichia coli, * Staohylococcus aureus, Enterobacter aerogenes or Pseudomonas aeruginosa, or of the fungi Candida albicans or Asgergillus niger or yeast Zvaosaccharomyces rouxii.
The inoculation should normally have a determined number of colonyforming units *.. (CFU) and the survival rate is determined by an aerobic plate count after a suitable incubation time, normally of 24 to 48 hours but longer for the fungi, the inoculated sample generally being plated out using the Surface Spread or Pour Plate techniques. The results are then compared with the regulatory specifications, which may for example demand a 99.9% decrease for the bacteria or yeast within a defined period of challenging the product sample with the micro-organism.
Although preservative efficacy testing is a formulation and regulatory necessity with well-established protocols as set out in the British, US and European Pharmacopoiea's for example, the traditional culture techniques for the testing are very time-consuming and laborious, requiring extensive hands-on testing with no potential for automation. These conventional techniques thus do not enable rapid,
L
high throughput screening and often are subcontracted by the manufacturer to an independent microbiological test laboratory with attendant further delays and costs.
In recent years attempts have been made to improve efficiency of the AET process by luminometric real time monitoring of AlP levels from test micro-organism cells as an indicator of the viable microbial biomass following challenge with a test substrate.
However, this has yet to prove a reliable replacement for the conventional cell culture approach.
It is a general objective of the present invention to provide an improved preservative efficacy testing system that overcomes one or more of the drawbacks of the conventional testing systems, providing substantial time and cost savings during development or production of the pharmaceuticals or cosmetics.
Summary of the Invention
According to a first aspect of the present invention there is provided a system for antimiôrobial preservative efficacy testing comprising a biosensor incorporating at : least one microbial strain selected from the group of micro-organisms comprising a regulatory recognised key micro-organism for preservative efficacy testing, the *** micro-organism being engineered to have a constitutive promoter linked to a lux or * ** luc gene cassette to produce a specific detectable signal reporting the viability of the * S S *** . . * micro-organism. *S.
The signal is effectively provided in real time at any point after initial inoculation and thus this system removes the long delay in result availability that is inherent in the art, whilst increasing the quantity and quality of data produced and decreasing the labour involved. The result is a rapid screening tool for evaluation and optimisation of preservative systems of pharmaceutical and cosmetic development compounds and formulations.
Particularly preferably the micro-organism is selected from the pharmacopoieal specified group of micro-organisms / regulatory recognised key micro-organisms comprising: Escherichia coil; Pseudomonas aeruginosa; Enterobacter aeroQenes; Candida albicans; Staphylococcus aureus:, Aspergillus niger; and Zvposaccharomyces rouxii.
The constitutive promoter is a promoter that normally enables a gene or operon to be constitutively expressed and which enables the Lux or Luc gene cassette to be constitutively expressed in the micro-organism (ie expressed continuously rather than only when induced).
The constitutive promoter is preferably selected from the group comprising: Ps (Lysyl-tRNA synthtase); P (spc ribosomal protein); Ptat ABCD (twin arginine translocase protein export system); (outer membrane lipoprotein); and Ppc (cold shock proteins).
In a particularly preferred embodiment the system is a provided as a kit and preferably is configured as a disposable self supporting multisample biosensor system that can by used in-house by customers.
In the art, bioluminescent bacteria usage as a means for toxicity testing has been practiced for over 20 years. Edinburgh Instruments, Merck Ltd, Azur Environmental and LUMlSmini have systems for the measurement of toxicity of waste water.
* .. Cybersense, Oxford UK have a multi sample bioluminescence based system, ROTAS, which utilizes naturally bioluminescent bacteria to assess toxicity in soil samples and Remedios Ltd use genetically-modified bioluminescent bacteria for lab- based analysis of contaminated land. Despite this, biosensor systems have not * ** * previously been proposed or developed for use in antimicrobial preservative efficacy testing. I. * * *
By conceiving and developing the biosensor system of the present invention for use ** ** in antimicrobial preservative efficacy testing we have immediately removed the lag time between taking of a sample and obtainment of results and enabled reliable real time responsive monitoring of formulation performance.
The biosensor system allows for automation, unlike current AET systems.
Furthermore, by speeding up the AET process, pharmaceutical and cosmetics companies will gain significant logistical, organisational, regulatory and financial benefits including reduced time to market for new formulations. The benefits of this new system may be extended to other end users including the food industry, amongst others.
Brief Description of the Drawings
A preferred embodiment of the present invention will now be more particularly described, by way of example, with reference to the accompanying drawings in which Figure 1 is a chart illustrating the construction of an example biosensor.
Detailed Description of Preferred Embodiments
The present invention will now be described by way of example only. These are not the only ways that the invention can be put into practice but they are the best ways currently known to the Applicant.
Firstly, a consititutive promoter for constitutive expression of the proposed reporter gene in the test micro-organism was selected and inserted into a suitable vector.
The test micro-organism of the preferred embodiment is one of the regulatory recognised key micro-organisms, Eschenchia coli, Staphylococcus aureus, Enterobacter aerogenes, Pseudomonas aerupinosa, Candida albicans. Aspergillus * ** gr or Zygosaccharomyces rouxii. * S * *.*S *.S* * *
*.** 20 For the illustrated test micro-organism, E coli ATCC 8739, the selected vector was ** the popular plasmid pBR322 and the promoter P was inserted between the EcoRl * .* * restriction cleavage site at the 4359 position on the plasmid and the BamHl S..
restriction cleavage site at the 375 position on the plasmid. * * *
*..:. 25 In the first example the promoter P was one of the promoters selected from: 1. P native downstream gene function: Lysyl-tRNA synthtase 2. P native downstream gene function: spc ribosomal protein operon promoter 3. P ABCD native downstream gene function: twin arginine translocase protein export system 4. P native downstream gene function: outer membrane lipoprotein 5. Pc native downstream gene function: cold shock proteins As shown in Figure 1, the preferred reporter gene cassette for use in the present invention is the Photorhabdus luminescens lux COABE gene cassette (Dewet 1985 PNAS 82:7870-7873) that is readily commercially available as carried in the plasmid pSB417. This is extracted from the commercial carrier plasmid by use of BamHl restriction endonuclease.
The pBR322 plasmid with the inserted constitutive promoter P was then also digested with BamHl so that the extracted lux CDABE gene cassette could then be ligated to the plasmid at the BamHl site and hence inserted into the pBR322 plasmid directly downstream of the promoter P. The thusformed construct was then transformed into the test micro-organism and plated out and screened for by its Ampicillin resistance and by its bioluminescence.
The engineered E coli. having the lux reporter gene and constitutive promoter was next incorporated into a prototype test kit for AET. This enabled real time assessment of the viable test microbe population following challenge with a sample product to be tested at any point in an AET investigation by measuring the light output with a PALcheck luminometer (Greer 2002 Luminescence 17: 43-74).
* *, The light output of stationary phase cultures of transformed bacterial cells correlated * * * against the traditional culture methods used for AET and against viable cell counts *a** enumerated by confocal microscopy demonstrates the efficacy of this new AET system and methodology. The stationary phase bacteria produce a light output * .* * relative to the population of viable stationary phase cells, indeed the output is more ** reliable than traditional plate count techniques, since Viable But Non Culturable (VBNC) cells are also detected by the method. Furthermore, bacteria that are actively growing can produce an enhanced signal, which may serve as a useful *.
early alarm for catastrophic failure of a formulation. This property along with the real time measurement offered by the biosensor will significantly cut the time required for formulations development.
Whereas traditional AET requires 28 days of monitoring and the actual logistical process takes approximately 45 days, if several iterations are needed AET becomes a significant component of formulation development time. By identifying failing formulations early the biosensor significantly cuts the total time required for formulation development, whilst throughput will be dramatically increased which will reduce the number of iterations required. Furthermore the number of man hours required per AET will be reduced and the removal of a 5 day lag time after each sample point will further cut the time required for AET.
Though the system is described above based upon use of a plasmid to introduce the reporter construct into the test micro-organism, in due course, rather than being plasmid-borne, the most preferred promoter-lux constructs will be integrated into the chromosome of the test bacterium by homologous recombination producing stable constructs as required for long term use and regulatory compliance.
Furthermore, for a commercial test kit the biosensor bacteria will be made up as a freeze dried product to produce a regulated, reliable, consistent product that can be reconstituted according to strict instructions by the end users. This will involve optimising batch and fed batch cultures to produce early stationary phase cultures, followed by washing and freeze-drying.
Although the exemplified constructs have a single reporter, two or more reporters may be incorporated if desired. Furthermore, the kit of the invention suitably comprises a battery of two or more of the test microorganisms, being different ones of the regulatory micro-organisms and/or having different promoters and/or reporter genes from each other to enable the user to carry out the required spread of tests for a given product type
I I.e I.
II * I.e I...
I p.CI
Claims (11)
- Claims 1. A system for antimicrobial preservative efficacy testingcomprising a biosensor incorporating at least one microbial strain selected from the group of micro- organisms comprising a regulatory recognised key micro-organism for preservative efficacy testing, the micro-organism being engineered to have a constitutive promoter linked to a lux or luc gene cassette to produce a specific detectable signal reporting the viability of the micro- organism.
- 2. A system for antimicrobial preservative efficacy testing as claimed in Claim 1, wherein the micro-organism is selected from the group of microorganisms comprising: Escherichia coli; Pseudomonas aeruginosa; Enterobacter aerogenes; Candida albicans; StaDhylococcus aureus: Aspergillus niger; and Zvgosaccharomvces rouxil.
- 3. A system for antimicrobial preservative efficacy testing as claimed in claim I or claim 2, wherein the constitutive promoter is selected from the group comprising: (Lysyl-tRNA synthtase); P8 (spc ribosomal protein); ABCD (twin arginine translocase protein export system); (outer membrane lipoprotein); and **,, P,c (cold shock proteins). * ..S H
- 4. A system for antimicrobial preservative efficacy testing as claimed in any preceding claim, wherein the system is provided as a kit.S
- 5. A system for antimicrobial preservative efficacy testing as claimed in Claim 4, wherein the kit comprises two or more said biosensors, each having a different key micro-organ ism.
- 6. A system for antimicrobial preservative efficacy testing as claimed in Claim 4 or 5, wherein the kit comprises two or more said biosensors, each having a different constitutive promoter.
- 7. A system for antimicrobial preservative efficacy testing comprising a biosensor incorporating at least one microbial strain selected from the group of micro- r - organisms comprising a regulatory recognised key micro-organism for preservative efficacy testing, the micro-organism being engineered to have a constitutive promoter linked to reporter gene to produce a specific detectable signal reporting the viability of the micro-organism.
- 8. A system for antimicrobial preservative efficacy testing as claimed in claim 7, wherein the system is provided as a kit and the kit comprises two or more said biosensors, each having a different reporter gene.
- 9. A system for antimicrobial preservative efficacy testing as claimed in any preceding claim and which is configured as a disposable self-supporting multi- sample biosensor system that can by used in-house by customers.
- 10. A system for antimicrobial preservative efficacy testing as claimed in any preceding claim, wherein the system is provided as a kit and the biosensor is in a freeze dried form.
- 11. A method of antimicrobial preservative efficacy testing comprising providing the system of any preceding claim and preparing a sample of product to be tested and introducing the biosensor of the system to the sample and observing any * ** change in luminescence of the biosensor.S S..S I. * ..* * S-S S
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB0615270A GB2429283B8 (en) | 2006-08-01 | 2006-08-01 | Preservative efficacy testing |
| PCT/GB2007/002899 WO2008015411A1 (en) | 2006-08-01 | 2007-07-31 | Preservative efficacy testing |
| DE112007001823T DE112007001823T5 (en) | 2006-08-01 | 2007-07-31 | Testing the effectiveness of preservatives |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB0615270A GB2429283B8 (en) | 2006-08-01 | 2006-08-01 | Preservative efficacy testing |
Publications (5)
| Publication Number | Publication Date |
|---|---|
| GB0615270D0 GB0615270D0 (en) | 2006-09-06 |
| GB2429283A true GB2429283A (en) | 2007-02-21 |
| GB2429283B GB2429283B (en) | 2007-08-08 |
| GB2429283B8 GB2429283B8 (en) | 2013-04-10 |
| GB2429283A8 GB2429283A8 (en) | 2013-04-10 |
Family
ID=37006555
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| GB0615270A Expired - Fee Related GB2429283B8 (en) | 2006-08-01 | 2006-08-01 | Preservative efficacy testing |
Country Status (3)
| Country | Link |
|---|---|
| DE (1) | DE112007001823T5 (en) |
| GB (1) | GB2429283B8 (en) |
| WO (1) | WO2008015411A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110286118A (en) * | 2019-06-18 | 2019-09-27 | 山东大学 | A method of antibiotic mechanism of action is determined using bioluminescence reporting system |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015178915A1 (en) * | 2014-05-22 | 2015-11-26 | Neogen Corporation | Automated preservative efficacy test method and device |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1990004037A1 (en) * | 1988-10-03 | 1990-04-19 | Genlux Forschungsgesellschaft Für Biologische Verfahren Mbh | Process for detecting and identifying toxic substances by means of cloned micro-organisms |
| WO1996010645A1 (en) * | 1994-10-03 | 1996-04-11 | Pathogenesis Corporation | Micobacterial reporter strains and uses thereof |
| WO1998049337A1 (en) * | 1997-05-01 | 1998-11-05 | Eastman Chemical Company | Bioluminescent reporter bacterium and methods for toxicity monitoring in biological wastewater treatment systems |
| US20060134724A1 (en) * | 2002-09-04 | 2006-06-22 | Kauppi Anna M | Method and probe for identifying bacterial virulence modifying agents, agents thus identifed, and their use |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002335979A (en) * | 2000-12-22 | 2002-11-26 | Pfizer Prod Inc | New bioluminescent assay and bacterial strain useful therein |
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2006
- 2006-08-01 GB GB0615270A patent/GB2429283B8/en not_active Expired - Fee Related
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2007
- 2007-07-31 WO PCT/GB2007/002899 patent/WO2008015411A1/en active Application Filing
- 2007-07-31 DE DE112007001823T patent/DE112007001823T5/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1990004037A1 (en) * | 1988-10-03 | 1990-04-19 | Genlux Forschungsgesellschaft Für Biologische Verfahren Mbh | Process for detecting and identifying toxic substances by means of cloned micro-organisms |
| WO1996010645A1 (en) * | 1994-10-03 | 1996-04-11 | Pathogenesis Corporation | Micobacterial reporter strains and uses thereof |
| WO1998049337A1 (en) * | 1997-05-01 | 1998-11-05 | Eastman Chemical Company | Bioluminescent reporter bacterium and methods for toxicity monitoring in biological wastewater treatment systems |
| US20060134724A1 (en) * | 2002-09-04 | 2006-06-22 | Kauppi Anna M | Method and probe for identifying bacterial virulence modifying agents, agents thus identifed, and their use |
Non-Patent Citations (3)
| Title |
|---|
| Antimicrob Agents Chemother; Vol 42, pp 1906-1910 (1998). Loimaranta et al. "Generation of bioluminescent streptococcus mutans and its usage in rapid analysis..." * |
| FEMS Microbiol Letts; Vol 199, pp 115-118 (2001). Parveen et al. "Biofilm culture of Pseudomonas aeruginosa expressing lux genes as a model to study susceptibility to antimicrobials" * |
| J Appl Bacteriol; Vol 75, pp 456-462 (1993). Connolly et al. "A study of the use of rapid methods for preservative efficacy testing of pharmaceuticals and cosmetics" * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110286118A (en) * | 2019-06-18 | 2019-09-27 | 山东大学 | A method of antibiotic mechanism of action is determined using bioluminescence reporting system |
| CN110286118B (en) * | 2019-06-18 | 2021-06-29 | 山东大学 | Method for judging antibiotic action mechanism by using bioluminescence report system |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2008015411A1 (en) | 2008-02-07 |
| GB2429283B (en) | 2007-08-08 |
| GB0615270D0 (en) | 2006-09-06 |
| DE112007001823T5 (en) | 2009-06-10 |
| GB2429283B8 (en) | 2013-04-10 |
| GB2429283A8 (en) | 2013-04-10 |
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