WO2019166752A1 - Système de divulgation pour détecter une contamination par un micro-organisme de surface - Google Patents

Système de divulgation pour détecter une contamination par un micro-organisme de surface Download PDF

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Publication number
WO2019166752A1
WO2019166752A1 PCT/GB2019/000031 GB2019000031W WO2019166752A1 WO 2019166752 A1 WO2019166752 A1 WO 2019166752A1 GB 2019000031 W GB2019000031 W GB 2019000031W WO 2019166752 A1 WO2019166752 A1 WO 2019166752A1
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WO
WIPO (PCT)
Prior art keywords
substrate
enzyme
disclosure
microorganism
gel
Prior art date
Application number
PCT/GB2019/000031
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English (en)
Inventor
Camilla Victoria ROBINSON
Alistair Hugh BISHOP
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The Secretary Of State For Defence
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Secretary Of State For Defence filed Critical The Secretary Of State For Defence
Publication of WO2019166752A1 publication Critical patent/WO2019166752A1/fr

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Classifications

    • 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
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • 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
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • 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
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • 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)
    • 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
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)

Definitions

  • the present invention is concerned with detecting surface microorganism contamination, especially a disclosure gel for visual detection of surface microorganism contamination, and more particularly a disclosure gel for detecting viable Bacillus anthracis spores.
  • Conventional detection and identification methods for microorganism contamination of surfaces include sensitive and specific genetic and antibody-based technologies such as polymerase chain reaction (PCR) and Enzyme Linked Immunosorbent Assay (ELISA).
  • PCR polymerase chain reaction
  • ELISA Enzyme Linked Immunosorbent Assay
  • microorganism contamination could be found in medical environments (e.g. MRSA contamination), industrial environments, be as a result of an environmental release (e.g.
  • Bacillus anthracis is a microorganism that could potentially be used in a hostile release due to its human pathogenicity and exceptional ability to survive in the environment through formation of metabolically inert spores.
  • the present invention thus generally aims to provide a simple means for detecting surface microorganism contamination, and in particular a simple means for detecting surface contamination by viable Bacillus anthracis spores.
  • the present invention provides a disclosure gel for detecting surface microorganism contamination, comprising a substrate for detecting an enzyme associated with the microorganism and a gelling agent, wherein the substrate is capable of generating a visually detectable signal, such as colour or fluorescence, upon digestion of the substrate by the enzyme.
  • the gelling agent is preferably hyaluronic acid or a derivative thereof.
  • the Applicant has conceived of an approach to detecting surface microorganism contamination which uses a substrate capable of generating a visually detectable signal to detect enzyme activity associated with a microorganism.
  • the visually detectable signal occurs upon digestion of the substrate by a specific enzyme.
  • the polysaccharide hyaluronic acid has a high moisture retention ability (providing a highly hydrated polymer formulation), which provides a particularly advantageous environment for an enzyme to function, as well as having a high viscoelasticity, combined with low immunogenicity and toxicity, making it a good candidate gel for widespread use in disclosure. It is also beneficial due to its ability to contain the spread of the disclosure gel, and thereby contain any contamination on the surface (i.e. it prevents the contamination from spreading across the surface).
  • the gelling agent hyaluronic acid preferably allows for the production of a hydrogel.
  • Hyaluronic acid thus provides a formulation which can be easily applied to a surface as a gel, but which also provides a good environment for the enzyme to function, to digest the substrate.
  • the hydrated polymer also allows for the properties of the formulation, such as rheological properties and viscoelasticity to be better controlled and optimised for use on surfaces, especially to co-locate at the site of the contamination, and to enable containment of the contamination and restrict disruption or movement of the contamination at or over the surface.
  • the microorganism may be any microorganism which a user may wish to test for and detect on a surface. Microorganisms all have associated enzymes (i.e.
  • the selected enzyme to target is preferably one that is secreted from the microorganism or is membrane-bound, as many substrates will be unable to penetrate the cytoplasmic membrane to be able to be digested by an enzyme at that location. The digestion of substrate by enzyme will be more likely to succeed and to succeed more rapidly where the enzyme is somehow associated with the surface of the microorganism.
  • the disclosure/detection could be for a group of microorganisms (where a specific enzyme is common to them all), or potentially could be specific to a particular species.
  • the enzyme is a glycosidase (also known as a glycoside hydrolase), which is an enzyme that catalyses the hydrolysis of glycosidic linkages, thereby degrading oligosaccharides and glycoconjugates.
  • glycosidase also known as a glycoside hydrolase
  • Glycosidases are a structurally diverse class of proteins, and thus can provide a good basis for distinguishing between species.
  • Glycosidases include such enzymes as a-D- glucosidase, b-D-glucosidase, a-D-galactosidase, b-D-galactosidase, a-L-arabinofuranosidase, N- acetyl ⁇ -glucosaminidase, and b-D-cellobiosidase.
  • the microorganism is Bacillus anthracis, and more particularly viable Bacillus anthracis spores.
  • Bacillus anthracis is a microorganism that could potentially be used in a hostile release due to its exceptional ability to survive in the environment through formation of metabolically inert spores, and consequently is a species of particular interest that needs to be detected simply, and preferably rapidly, on surfaces.
  • a number of enzymes have been identified in the outermost layer of the B. anthracis spore; the exosporium. These include alanine racemase and inosine-uridine-preferring nucleoside hydrolase, which interact with the environment and may function to degrade the germinants L-alanine and inosine, respectively, to suppress germination in unfavourable conditions.
  • Glycosidase activity is also present in viable Bacillus anthracis spores, in the exosporium, especially or-glucosidase activity.
  • the general role of a-glucosidases is to catalyze the hydrolysis of terminal, non-reducing, 1, 4-1 inked a-D-glucose residues, with oligosaccharides being the favoured substrate.
  • Glucosidase activity is poorly characterised in B. anthracis. However, it is known that a-D-glucosidase activity but not b-D-glucosidase activity is present in the organism.
  • Such enzyme as a-glucosidase is well suited to enzyme-activated disclosure and detection of viable spores since the enzyme is extracellular, residing in the exosporium.
  • the substrate should be a reagent that can be digested by the chosen enzyme to generate a visible signal, such as colour or fluorescence.
  • Substrates for glycosidases are likely to include glycans (monosaccharides or oligosaccharides) and glycosides, modified by fluorogenic or chromogenic tags, such that on digestion fluorescence or colour is generated through release of the tag, or modified tag.
  • the substrate may comprise 4-methylumbelliferyl derivatives, 7-amido-4-methyl-coumarin derivatives, or diacetylfluorescein derivatives, which substrates are capable of reacting with, or capable of being reacted upon by, a selected enzyme to produce a fluorescent product.
  • the substrate may comprise 5-bromo-4-chloro-3-indolyl derivatives, nitrophenyl derivatives, indoxyl derivatives, or phenolphtalein derivatives, which substrates are capable of reacting with, or capable of being reacted upon by, said enzyme to produce a coloured product.
  • the substrate could be 4-methylumbelliferyl-a-D- glucopyranoside, which embodiment is suitable for detecting/disclosing viable Bacillus anthracis spores.
  • This embodiment is particularly effective where the gelling agent comprises hyaluronic acid.
  • This embodiment has resulted in an easily applied gel formulation for wide-area disclosure of the location and extent of surface contamination with viable Bacillus anthracis spores. The gel remains hydrated for a sufficient time for -glucosidase activity present in B. anthracis spores to cleave the substrate and release the visual signal: the fluorescent product methylumbelliferone.
  • the presence of B. anthracis spores could be disclosed at 5 x 10 4 CFU both visually and using fluorescence detection equipment, with good specificity.
  • the disclosure gel may additionally comprise reagents to optimise or enhance the enzyme activity and digestion.
  • a-glucosidase activity may be enhanced by addition of maltose or sucrose to improve the visible signal generated.
  • the disclosure gel may additionally comprise germinants to enhance the activity of the enzyme or disclosure gel (e.g. L-alanine; inosine), which could be through allowing growth/maintenance of the microorganism of interest.
  • germinants e.g. L-alanine; inosine
  • the disclosure gel comprises, or consists, of 4- methylumbelliferyl-a-D-glucopyranoside maltose, L-alanine, inosine and hyaluronic acid in a NaCI solution.
  • the disclosure gel of the first aspect provides a simple, rapid, visual response to the presence of a microorganism on a surface.
  • the present invention provides a method for detecting surface microorganism contamination comprising a. contacting with a suspect surface a disclosure gel comprising a substrate for detecting an enzyme associated with said microorganism, wherein said substrate is capable of generating a visually detectable signal, such as colour or fluorescence, upon digestion of the substrate by the enzyme; b. incubating the disclosure gel with the suspect surface under reaction conditions suitable for digestion of the substrate by the enzyme for a pre-defined period of time; c.
  • the disclosure gel advantageously comprises hyaluronic acid, or a derivative thereof.
  • Figure 1 shows fluorescence from the disclosure gel after a 3 h incubation with intact washed spores at 1 x 10 7 CFU ml '1 (A), autoclaved spores at lx 10 7 CFU ml ' ⁇ B), and no spores (C). Fluorescence was visualised after addition of NaOH (250 mmol G 1 final concentration) under UV light with a 485 - 655 nm emission filter;
  • Figure 2 shows visual fluorescence output from washed intact B. anthracis Sterne spores in suspension (A), after drying onto the well surface (B) and autoclaved spores (C) after 3 h at 25 °C. Fluorescence was visualised after addition of NaOH (250 mmol G 1 final concentration) under UV light with a 485 - 655 nm emission filter; and
  • Figure 3 represents the effect of soil on observation of fluorescence.
  • Each reaction contained 5x10 s CFU B. anthracis spores in addition to soil suspension in PBS or PBS alone (No soil) and disclosure gel in a total volume of 50 pi.
  • RFU equates to average fluorescence output above the disclosure gel background fluorescence after 3 h incubation in 16 experiments. Error bars show standard deviation from the mean.
  • Methylumbelliferyl -a-D-glucopyranoside was combined with hyaluronic acid to produce a gel that could be applied to a surface as a coating.
  • a disclosure gel has been developed to visually alert to the presence of B. anthracis spores on a surface.
  • the polysaccharide hyaluronic acid was selected due to its high moisture retention ability and viscoelasticity, combined with low immunogenicity and toxicity, making it a potentially suitable gel for widespread use in disclosure.
  • a range of hyaluronic acid concentrations were tested for optimal rheological properties.
  • a range of buffers and sugars were tested by inclusion in the formulation.
  • Enzyme activity was also tested over a pH range of 5 - 7.4 to determine the optimal pH conditions. Fluorescence output was reduced with PBS, acetate buffer and phosphate citrate buffer (all pH 7) compared with 150 mmol G 1 NaCI, and a pH of 7 to 7.4 was found to be optimal for each buffer.
  • Maltose and to a lesser extent, sucrose, improved fluorescence output from spores, up to a final concentration of 100 mmol G 1 sugar (data not shown). Maltose was therefore included in the formulation as an inducer of a-glucosidase activity.
  • the disclosure gel was prepared with 1 mmol I '1 4-methylumbelliferyl-a-D-glucopyranoside (a-MUG), 100 mmol I 1 maltose, 100 mmol I 1 L-alanine, 10 mmol G 1 inosine and 0.7 % (w/v) hyaluronic acid (mol. wt. 750,000-1,000,000) in 150 mmol I '1 NaCI. Water retention of the hyaluronic acid-based disclosure gel was measured by weight over time.
  • a-MUG 4-methylumbelliferyl-a-D-glucopyranoside
  • anthracis spores was measured by detection of the fluorescent product 4-MU released during a 3 h incubation period in the presence of a-MUG. This method demonstrated visually detectable enzyme activity from intact spores against a background of dead (autoclaved) spores.
  • the addition of freshly prepared amino acid germinants (100 mmol G 1 L-alanine and 10 mmol I 1 inosine) led to an increase in relative fluorescence (RFU) compared to the formulation without germinants for spores at a final concentration of lx 10 7 CFU ml '1 . From eight experiments the average increase was 62 % with a standard deviation of ⁇ 6 %.
  • the increase in a-glucosidase activity may be due to new enzyme synthesis as a result of initiating germination or release of a- glucosidases from within the inner layers of the spore.
  • Table 1 Relative fluorescence output from B. anthracis Sterne spores in suspension or dried onto a surface after incubation with disclosure gel for 3 h at 25 °C. RFU equates to average fluorescence output above the disclosure gel background fluorescence from eight individual experiments.
  • the disclosure gel was tested against a panel of Bacillus species and other environmental or skin- associated bacterial species to determine its exclusivity. As expected, very little a-g!ucosidase activity was detected from the intact vegetative cells tested as a-MUG is unable to cross into the cytoplasm, which is where most known a-glucosidases are present. The lack of medium to support growth would also have reduced the incidence of any extracellular a-glucosidases being produced. Bacillus thuringiensis HD-1 Cry , but not the other environmental B. thuringiensis strains tested, had a similar level of a-glucosidase activity to B. anthracis in the disclosure gel (Table 2).
  • Table 2 Relative a-glucosidase activity in washed spores ⁇ Bacillus spp.) or vegetative cells (non- Bacillus spp.) after incubation with disclosure gel for 3 h at 25 °C.
  • Bacillus lichenlformis spores also displayed notable -MUG hydrolysis, albeit at a lower rate than both B. anthracis and B. thuringiensis HD-1 Cry ' . Although a spore-associated a-glucosidase has not been described in the literature, B. licheniformis is known to produce both cytoplasmic and extracellular a-glucosidases so this activity is not unexpected.
  • Soil and skin contact were regarded as likely contaminants of surfaces suspected to have been exposed to bacterial agents of concern. They were, therefore, assessed for their potential interference on a-glucosidase activity from B. anthracis spores using the disclosure gel.
  • Table 4 Fluorescence from disclosure gel in plates that had or had not been handled for 5 min, with and without the addition of lxlO 7 cfu ml '1 B. anthracis spores. The results were obtained from 6 replicate experiments. Standard deviation from the mean is shown in parentheses.
  • a polymer-based gel formulation has been developed and optimised to successfully allow visual determination of the presence and location of surface contamination with live B. anthracis spores.
  • the results show that a-glucosidase activity present in intact B. anthracis spores can be used to cleave the synthetic substrate a-MUG to produce a fluorescent response which can be visualised under UV light within a few hours.
  • the disclosure gel does not respond to a range of common environmental Bacillus or other bacterial species under the given conditions, affording a good degree of specificity for initial assessment of equipment following a suspected release of biological agent.
  • a number of strains tested are known to produce a-glucosidases; in other Bacillus species a-glucosidases have largely been reported in cytoplasmic or extracellular locations and Staphylococcus aureus (MalA) and Pseudomonas fluorescens produce cytoplasmic a-glucosidases. These and most other strains tested produced little or no fluorescence in response to the disclosure gel.

Abstract

La présente invention concerne un gel de divulgation pour détecter une contamination par un micro-organisme de surface, en particulier un gel de divulgation pour détecter des spores de bacillus anthracis revivifiables. La présente invention porte également sur des méthodes d'utilisation associées.
PCT/GB2019/000031 2018-03-02 2019-02-22 Système de divulgation pour détecter une contamination par un micro-organisme de surface WO2019166752A1 (fr)

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GB1803437.1 2018-03-02
GBGB1803437.1A GB201803437D0 (en) 2018-03-02 2018-03-02 A disclosure system for detecting surface microorganism contamination

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ATE236176T1 (de) * 2000-12-27 2003-04-15 Biosynth Ag Neue substrate zur detektion von mikrobiellen metaboliten
US7309580B2 (en) * 2002-07-05 2007-12-18 R&F Products, Inc. Chromogenic plating medium for the rapid presumptive identification of Bacillus anthrasis, Bacillus cereus, and Bacillus thuringiensis
US7749724B2 (en) * 2005-07-05 2010-07-06 Washington State University Fluorogenic selective and differential medium for isolation of Enterobacter sakazakii
PT2433138T (pt) * 2009-05-18 2016-12-19 Technische Universität Graz Método para detectar infecções numa ferida
EP2785404B1 (fr) * 2011-11-28 2020-09-02 Hyprotek, Inc. Dispositifs de cathéter et techniques

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020115131A1 (en) * 1999-02-22 2002-08-22 3M Innovative Properties Company Rapid readout sterilization indicator for liquid peracetic acid sterilization procedures
US20160102335A1 (en) * 2014-10-13 2016-04-14 American Sterilizer Company Biological indicator
WO2016156773A1 (fr) * 2015-03-30 2016-10-06 Microbiosensor Limited Dispositifs de détection microbienne

Non-Patent Citations (1)

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Title
C V ROBINSON ET AL: "A disclosure gel for visual detection of live Bacillus anthracis spores", JOURNAL OF APPLIED MICROBIOLOGY., 18 February 2019 (2019-02-18), GB, XP055580343, ISSN: 1364-5072, DOI: 10.1111/jam.14226 *

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GB2572482A (en) 2019-10-02
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