WO2008015411A1 - Preservative efficacy testing - Google Patents

Preservative efficacy testing Download PDF

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Publication number
WO2008015411A1
WO2008015411A1 PCT/GB2007/002899 GB2007002899W WO2008015411A1 WO 2008015411 A1 WO2008015411 A1 WO 2008015411A1 GB 2007002899 W GB2007002899 W GB 2007002899W WO 2008015411 A1 WO2008015411 A1 WO 2008015411A1
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WIPO (PCT)
Prior art keywords
micro
preservative efficacy
efficacy testing
organism
antimicrobial preservative
Prior art date
Application number
PCT/GB2007/002899
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French (fr)
Inventor
David Naseby
Original Assignee
University Of Hertfordshire
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Publication date
Application filed by University Of Hertfordshire filed Critical University Of Hertfordshire
Priority to DE112007001823T priority Critical patent/DE112007001823T5/en
Publication of WO2008015411A1 publication Critical patent/WO2008015411A1/en

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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
    • 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

Definitions

  • the present invention relates to a system and method for antimicrobial preservative efficacy testing (AET or PET) for the pharmaceutical and cosmetic/ toiletry industries.
  • AET antimicrobial preservative efficacy testing
  • 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 inoculated with a microbial suspension of a population of a regulatory recognised key micro-organism, normally being one of the bacteria Escherichia coli. Staphylococcus aureus. Enterobacter aeroqenes or Pseudomonas aeruginosa, or of the fungi Candida albicans or Aspergillus niqer or yeast Zygosaccharomyces rouxii.
  • the inoculation should normally have a determined number of colony-forming 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.
  • CFU colony-forming units
  • 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.
  • a system for antimicrobial 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 signai reporting the viability of the micro-organism.
  • 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.
  • the- micro-organism is selected from the pharmacopoieai specified group of micro-organisms / regulatory recognised key micro-organisms comprising: Escherichia coli; Pseudomonas aeruginosa; E ⁇ terobacter aero ⁇ enes: Candida albicans; Staphylococcus aureus:. Aspergillus niqer: and Zvgosaccharomvces rouxii.
  • the constitutive promoter is a promoter that normally enables a gene or operon to be constitutiveiy expressed and which enables the Lux or Luc gene cassette to be constitutiveiy expressed in the micro-organism (ie expressed continuously rather than only when induced).
  • the constitutive promoter is preferably selected from the group comprising: Pi ysS (Lysyl-tRNA synthtase); P spc (spc ribosomal protein); P ⁇ A BCD (twin arginine translocase protein export system); P ⁇ pp (outer membrane lipoprotein); and Pcspc ( cold shock proteins).
  • the system is a provided as a Wt and preferably is configured as a disposable self supporting multisample biosensor system that can by used in-house by customers.
  • bioluminescent bacteria usage as a means for toxicity testing has been practiced for over 20 years. Edinburgh instruments, Merck Ltd, Azur Environmental and LUMISmini 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.
  • 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.
  • Figure 1 is a chart illustrating the construction of an example biosensor.
  • test micro-organism of the preferred embodiment is one of the regulatory recognised key micro-organisms, Escherichia coli. Staphylococcus aureus. Enterobacter aerogenes, Pseudomonas aeruginosa. Candida albicans. Aspergillus niger or Zygosaccharomyces rouxii.
  • E coli ATCC 8739 E coli ATCC 8739
  • the selected vector was the popular plasmid pBR322 and the promoter P was inserted between the EcoR1 restriction cleavage site at the 4359 position on the plasmid and the BamH1 restriction cleavage site at the 375 position on the plasmid.
  • the promoter P was one of the promoters selected from:
  • Piy s s native downstream gene function Lysyl-tRNA synthtase
  • P spc native downstream gene function spc ribosomal protein operon promoter
  • Pat AB G D native downstream gene function twin arginine transiocase protein export system
  • the preferred reporter gene cassette for use in the present invention is the Photorhabdus luminescens lux CDABE 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 BamH1 restriction endo ⁇ uclease.
  • the pBR322 plasmid with the inserted constitutive promoter P was then also digested with BamH1 so that the extracted lux CDABE gene cassette could then be ligated to the plasmid at the BamH1 site and hence inserted into the pBR322 piasmid 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 bioiuminescence.
  • 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 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 ceils, indeed the output is more reliable than traditional plate count techniques, since Viable But Non Culturable (VBNC) ceils are also detected by the method.
  • VBNC Non Culturable
  • 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.
  • 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.
  • the kit of the invention suitably comprises a battery of two or more of the test micro-organisms, 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

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Toxicology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Virology (AREA)
  • Cell Biology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Food Science & Technology (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The present invention concerns improvements in and relating to antimicrobial preservative efficacy testing (AET) and in a first aspect provides a system for antimicrobial 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 micro-organism.

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 inoculated with a microbial suspension of a population of a regulatory recognised key micro-organism, normally being one of the bacteria Escherichia coli. Staphylococcus aureus. Enterobacter aeroqenes or Pseudomonas aeruginosa, or of the fungi Candida albicans or Aspergillus niqer or yeast Zygosaccharomyces rouxii. The inoculation should normally have a determined number of colony-forming 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, 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 byluminometric real time monitoring of ATP 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 ofthe 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 antimicrobial 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 signai reporting the viability of the micro-organism.
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 pharmacopoieai specified group of micro-organisms / regulatory recognised key micro-organisms comprising: Escherichia coli; Pseudomonas aeruginosa; Eπterobacter aeroαenes: Candida albicans; Staphylococcus aureus:. Aspergillus niqer: and Zvgosaccharomvces rouxii. The constitutive promoter is a promoter that normally enables a gene or operon to be constitutiveiy expressed and which enables the Lux or Luc gene cassette to be constitutiveiy expressed in the micro-organism (ie expressed continuously rather than only when induced).
The constitutive promoter is preferably selected from the group comprising: PiysS (Lysyl-tRNA synthtase); Pspc (spc ribosomal protein); P^ ABCD (twin arginine translocase protein export system); Pιpp (outer membrane lipoprotein); and Pcspc ( cold shock proteins).
In a particularly preferred embodiment the system is a provided as a Wt 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 LUMISmini 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.
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, Escherichia coli. Staphylococcus aureus. Enterobacter aerogenes, Pseudomonas aeruginosa. Candida albicans. Aspergillus niger or Zygosaccharomyces rouxii.
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 EcoR1 restriction cleavage site at the 4359 position on the plasmid and the BamH1 restriction cleavage site at the 375 position on the plasmid.
In the first example the promoter P was one of the promoters selected from:
1. Piyss native downstream gene function: Lysyl-tRNA synthtase
2. Pspc native downstream gene function: spc ribosomal protein operon promoter
3. Pat ABGD native downstream gene function: twin arginine transiocase protein export system
4. Pipp native downstream gene function: outer membrane lipoprotein
5. Pcspc 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 CDABE 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 BamH1 restriction endoπuclease.
The pBR322 plasmid with the inserted constitutive promoter P was then also digested with BamH1 so that the extracted lux CDABE gene cassette could then be ligated to the plasmid at the BamH1 site and hence inserted into the pBR322 piasmid 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 bioiuminescence.
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 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 ceils, indeed the output is more reliable than traditional plate count techniques, since Viable But Non Culturable (VBNC) ceils 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 wiii 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 micro-organisms, 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

Claims

Claims
1. A system for antimicrobial preservative efficacy testing comprising a biosensor incorporating at least one microbial strain selected from the group of microorganisms 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 micro-organisms comprising: Escherichia coli; Pseudomonas aeruginosa; Enterobacter aerogenes; Candida albicans; Staphylococcus aureus; Aspergillus niqer; and Zvgosaccharomvces rouxii.
3. A system for antimicrobial preservative efficacy testing as claimed in claim 1 or claim 2, wherein the constitutive promoter is selected from the group comprising: Piyss (Lysyi-tRNA synthtase); Pspc (spc ribosomal protein); Ptat ABCD (twin arginine translocase protein export system); Pιpp (outer membrane lipoprotein); and Pcspc ( cold shock proteins).
4. A system for antimicrobial preservative efficacy testing as claimed in any preceding claim, wherein the system is provided as a kit.
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-organism.
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- 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.
PCT/GB2007/002899 2006-08-01 2007-07-31 Preservative efficacy testing WO2008015411A1 (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
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
US9625479B1 (en) 2014-05-22 2017-04-18 Neogen Corporation Automated preservative efficacy test method and device

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