WO2019166752A1

WO2019166752A1 - A disclosure system for detecting surface microorganism contamination

Info

Publication number: WO2019166752A1
Application number: PCT/GB2019/000031
Authority: WO
Inventors: Camilla Victoria ROBINSON; Alistair Hugh BISHOP
Original assignee: UK Secretary of State for Defence
Current assignee: UK Secretary of State for Defence
Priority date: 2018-03-02
Filing date: 2019-02-22
Publication date: 2019-09-06
Anticipated expiration: 2020-09-02
Also published as: GB201803437D0; GB201902682D0; GB2572482A

Abstract

The present invention provides a disclosure gel for detecting surface microorganism contamination, especially a disclosure gel for detecting viable bacillus anthracis spores. The present invention also provides methods of use thereof.

Description

A Disclosure System for Detecting Surface Microorganism Contamination

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). Two problems encountered with such methods are surface sampling and differentiation between living and dead bacteria. Traditional plate count methods give an indication of viability but are time-consuming and do not avoid the issue of surface sampling, which may be of particular concern given the likelihood of non-uniform agent deposition on equipment surfaces.

Such microorganism contamination could be found in medical environments (e.g. MRSA contamination), industrial environments, be as a result of an environmental release (e.g.

agricultural/farming) or could be as a result of a hostile release of a microorganism. 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.

A simple approach to detect and locate living/viable microorganisms on a surface is required.

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 Applicant has recognised that enzymes associated with a living cell could be used to trigger the disclosure, detection or identification of a microorganism, and that this could potentially be specific depending on the choice of enzyme and target microorganism. Accordingly, in a first aspect, 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. present within their cells, or on or near to their cell surfaces) which could be used in conjunction with a specific substrate to disclose/detect the presence of the microorganism. 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.

In one embodiment, 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. 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.

In one embodiment of the invention 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.

The natural course of colonisation and infection of a mammal are also important stages in the lifecycle of the pathogen. To survive the onslaught of the immune system, the first line of defence is again through enzymes within the exosporium. Superoxide dismutase and arginase are thought to play important roles in controlling intracellular survival in macrophages through protection from oxidative injury and inhibition of nitric oxide synthesis within a macrophage. Activities of superoxide dismutase, inosine hydrolase and arginase have been recorded in intact endospores and semi- purified exosporium. 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.

In one embodiment for targeting a a-glucosidase, 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 10⁴ 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. For example, 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.

In one embodiment of the first aspect, 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. In a second aspect, 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. interrogating the suspect surface for the visually detectable signal, wherein the visually detectable signal is indicative of the presence or surface microorganism contamination by said microorganism. The disclosure gel advantageously comprises hyaluronic acid, or a derivative thereof. The present invention will now be described with reference to the following non-limiting examples and drawings in which

Figure 1 shows fluorescence from the disclosure gel after a 3 h incubation with intact washed spores at 1 x 10⁷ CFU ml^'1 (A), autoclaved spores at lx 10⁷ CFU ml^'^B), and no spores (C). Fluorescence was visualised after addition of NaOH (250 mmol G¹ 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 G¹ 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.

Example - A Disclosure Gel for Visual Detection of Viable Bacillus anthracls Spores

Methylumbelliferyl -a-D-glucopyranoside was combined with hyaluronic acid to produce a gel that could be applied to a surface as a coating. By combining a substrate to detect enzyme activity in viable spores with a highly hydrated polymer formulation which can easily be applied 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.

Disclosure gel development, preparation and testing

A range of hyaluronic acid concentrations were tested for optimal rheological properties. To evaluate the effect of various inducers and ions on cr-glucosidase activity from intact B. anthracls spores, 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 G¹ 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 G¹ 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^'14-methylumbelliferyl-a-D-glucopyranoside (a-MUG), 100 mmol I ¹ maltose, 100 mmol I ¹ L-alanine, 10 mmol G¹ 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.

Three individual 1 ml samples of the gel were pipetted onto glass slides and weighed. The slides were then incubated at 20 °C, 85 % relative humidity on an open tray. Weights were measured again after 3 and 6 hours incubation. Water retention by the disclosure gel after 3 h incubation was 86 % ± 0.2 % and after 6 h was 69 % ± 0.3 %.

To determine the effect of the formulation on germination of Bacillus anthracis Sterne spores, a final concentration of 1 x 10⁷ CFU ml^'1 spores was added to the disclosure gel and incubated for 3 h at 25 °C. The percentage of germinating spores, as determined by a change to phase dark, was determined through visual inspection at 400 x magnification and compared to that of spores with germinants (100 mmol G¹ L-alanine, 10 mmol G¹ inosine) alone added. The germination rate for B. anthracis spores in the disclosure gel was 98 %.

Determination of a-glucosidase activity using the disclosure gel

Five microlitres of spore stock (at 1 x 10^s CFU ml^'1) or washed vegetative bacterial suspension were added to 45 mI of the disclosure gel in a flat-bottomed black 96-well microplate (Nunc). Assay reactions were incubated at 25 °C for 3 h unless otherwise stated. To stop the reaction and obtain optimal fluorescence, 50 pi of 0.5 mol I^'1 NaOFI were added to each well. Fluorescence was measured at an excitation/emission wavelengths of 365/455 nm, using an Infinite M200 Pro fluorescence microplate reader (Tecan). Wells without spores (blank) and with autoclaved (dead) spores were used as controls. Relative fluorescence was calculated by subtracting the fluorescence level of the blank (reagents only) wells from the test wells. Each plate included a calibration curve of 4-methylumbelliferone (4-MU) in dH₂0, ranging from 0 to 50 pmol in a total volume of 100 mI, prepared in duplicate. The amount of 4-MU released in each test well was calculated using the slope of the calibration curve. Visual observation of the fluorescence output was carried out under a UV light source with a 485 - 655 nm emission filter. Having regard to Figure 1, enzyme activity associated with intact, washed B. 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 G¹ 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 10⁷ 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.

Where spores dried onto the plate surface were to be tested, 5 mI spore stock were pipetted into individual wells and the plates were left uncovered in a microbiological cabinet overnight. Disclosure gel was added on top of the dried spores and incubated for 3 h at 25 °C before addition of 0.5 mol G¹ NaOH and recording of fluorescence. Having regard to Figure 2, for spores both in suspension and dried onto a surface (0.32 cm² area), 5 x 10⁴ CFU could be consistently detected above background fluorescence both visually and using fluorescence detection equipment (Table 1). This equates to approximately 1 - 20 times the predicted human infectious dose (ID₅₀) of spores by the inhalational route.

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.

Standard deviation from the mean is shown in parentheses.

Spores that had been left to dry on a surface before addition of the gel produced slightly lower average fluorescence output than spores in suspension, which would be expected due to lower accessibility to the substrate. Autoclaved spores could not be distinguished from background fluorescence.

Bacterial exclusivity of the disclosure formulation

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.

Effect of soil and skin contamination on a-glucosidase activity

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.

Garden soil (lime-rich loamy soil) was suspended in sterile PBS to a concentration of 10 mg ml ¹. Ten- fold serial dilutions of the soil suspension were made both with and without addition of B. anthracis spore stock (1 x 10^s CFU ml^'1 final concentration) and 5 mI of each dilution were tested for fluorescence output after 3 h incubation with disclosure gel. Sterile PBS was used as a negative control sample.

For skin contamination, this was crudely tested using polystyrene 6-well plates (Corning). Two individuals using unwashed hands touched the surface of each well within two 6-well plates multiple times for 5 minutes. To one plate that had been handled by each individual, B. anthracis spore stock at lxlO⁹ CFU ml^'1 was added in five drops, 2 mI each, around the surface of each well. The other plate handled by each individual had sterile water added in the same manner. Sterile, untouched plates were inoculated in the same way with spore stock or water for use as comparative controls. All plates had 990 mI disclosure gel added to the centre of each well, which spread to cover the surface. After 3 h incubation at 25 °C and addition of 1 ml NaOH (0.5 mol I ¹) to each well, 100 mI samples were transferred from each well into a black 96-well plate before fluorescence was measured.

Statistical analysis was carried out using t tests with the null hypothesis being that there would be no difference between assays carried out in the presence of soil or skin contact and those carried out in their absence. Differences with P values of <0.05 were considered statistically significant. Having regard to Figure 3, garden soil suspension at a final concentration of 1 mg ml^'1 in the gel showed considerable interference with fluorescence output, producing a significantly lower average RFU than the 'no soil' control (p=0.006), with wide variation. No difference was observed between the other soil dilutions and the 'no soil' control. The soil suspension was confirmed as pH 7. The interference with fluorescence may therefore be due to the organic matter present within the soil.

As an indication of the levels of B. cereus or B. thuringiensis naturally occurring in the soil sample, the selective medium Brilliance Bacillus cereus agar was used to spread culture aliquots of the soil suspension at 10 mg ml ¹. An average of 140 blue colonies were observed per 1 ml of soil suspension, equating to fewer than one CFU per reaction well in the assay set-up. As expected from this result, no a- UG hydrolysis was observed from soil suspension alone in the disclosure gel at any concentration, suggesting that false positive responses to disclosure gel application would not be caused by soil contamination with this soil type.

Skin contact on a surface prior to contamination with spores and application of disclosure gel resulted in a statistically significant reduction in fluorescence (p=< 0.0001). The fluorescence output was reduced by almost half over the course of the 3 hour incubation (Table 4). A small but not statistically significant (p= 0.08) amount of fluorescence was observed on surfaces that had been handled but not exposed to B. anthracis spores. This is not sufficient to pose a risk of a visual false positive reaction from the 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 lxlO⁷ 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. Therefore, their presence on surfaces is unlikely to interfere with the visual output of the formulation or produce false positive signals unless present at very high inoculum levels, in the case of some Bacillus spp. spores, or in an active growth stage due to alternative nutrient sources; the lack of essential nutrients in the disclosure gel ensures it is not conducive to active metabolism and undesirable cell growth.

Claims

1. 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, and the gelling agent is hyaluronic acid, or a derivative thereof.

2. A disclosure gel according to Claim 1, wherein the microorganism is viable Bacillus anthracis spores.

3. A disclosure gel according to Claim 1 or Claim 2, wherein the enzyme is a glycosidase.

4. A disclosure gel according to Claim 3, wherein the glycosidase is a a-glucosidase.

5. A disclosure gel according to Claim 4, wherein the substrate is 4-methylumbelliferyl-a-D- glucopyranoside.

6. 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 and hyaluronic acid, or a derivative thereof, 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. interrogating the suspect surface for the visually detectable signal, wherein the visually detectable signal is indicative of the presence or surface microorganism contamination by said microorganism.

7. Use of a substrate in a gel comprising hyaluronic acid, or a derivative thereof, for detecting an enzyme associated with a microorganism in detecting surface microorganism contamination, wherein the substrate is capable of generating a visually detectable signal, such as colour or fluorescence, upon digestion of the substrate by the enzyme.