WO2019177541A1 - A dye suitable for use in the differentiation of viable and non-viable bacteria - Google Patents

Abstract

There is provided a compound of formula I, having the structure: wherein R1 to R6, and X have meanings given in the description. Also disclosed herein is a method of detecting and/or quantitating the presence of metabolically active microorganisms in a test sample using said compound.

Description

A Dye suitable for use in the Differentiation of Viable and Non-viable Bacteria

Field of Invention

The current invention relates to the use of compounds in PCR methods to detect and/or quantitate the presence of metabolically active microorganisms in a test sample.

Background

The listing or discussion of a prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common genera! knowledge.

A major disadvantage of PCR-based methods is that they do not discriminate viable from non- viable cells, which can lead to an overestimation of the number of viable cells (Nocker, A., et al. 2006 J Microbiol Methods. 67, 310-320 and Rudi, et al. 2005. Appl Environ Microbiol. 71 , 1018-1024). Recently, researchers have considered combining ethidium monoazide (EMA) or propidium monoazide (PMA) with quantitative real-time PCR (qPCR) to be a promising method to distinguish between viable and non-viable cells (ibid). Both EMA and PMA-PCR assays have been successfully applied to discriminate viable from dead bacteria in such diverse samples as water, food, air and clinical settings (Bae, S., and Wuertz, S., 2009. Water Res. 43, 4850-4859; Li, B., and Chen, J.Q., 2013. BMC Microbiol. 13, 273; Miotto, et al 2012. Eur Respir J. 39, 1269-1271 ; and Rogers, G.B., et al 2008. Diagn Microbiol Infect Dis. 62, 133- 141). These intercalating dyes have a photo-inducible azide group that covalently binds to DNA and can only penetrate membrane-compromised cells. When put in visible light, the photoreactive azide group on the PMA (or EMA) is converted to a highly reactive nitrene radical, which readily reacts with any hydrocarbon moiety of DNA. It forms a stable covalent nitrogen-carbon bond, which interferes with PCR amplification. However, a fundamental drawback of using DNA-intercalating dyes is that they function based on membrane integrity as a viability criterion, rather than the physiological states of the cells being evaluated (e.g., metabolically active or active but non-culturable cells, see Nocker, A., and Camper, A.K., 2009. Ferns Microbiology Letters. 291 , 137-142). Assessing bacterial physiology using either an EMA- or PMA-(q) PCR remains a challenge. Thus, EMA or PMA-(q) PCR often overestimates the viable cell population when under inactivation conditions such as UV treatment or antibiotic treatment (Kobayashi, H., et a/2010. J Orthop Res. 28, 1245-1251 and Nocker, A., Journal of Microbiological Methods. 70, 252-260). Thus, there remains a need for new materials that can assist in overcoming the problems discussed above for PCR-related methods.

Summary of Invention

In a first aspect of the invention, there is provided a compound of formula I:

where:

R¹ is C1-6 alkyl and R² is H, or R¹ and R² together with the atoms that they are attached to form a 5- to 8-membered heterocyclic ring;

R³ represents H, Y(CH₂)nN₃, or 0C(=0)CH₂N₃;

R⁴ represents Y(CH₂)_nN₃, (CH₂)_mN⁺(R⁷)₃ or aryl, where the aryl group is unsubstituted or substituted by halo, Ci-₆ alkyl or Y(CH₂)_nN₃;

R⁵ represents H, OR⁸ or Y(CH₂)_nN₃;

R⁶ represents H or OR⁸;

R⁷ and R⁸ independently represent at each occurrence C1-6 alkyl;

Y represents NR⁹ or O;

X represents one or more counter ions to balance the positive charge of the organic molecule; R⁹ represents H or C1 -6 alkyl;

n is a number from 2 to 10;

m is a number from 2 to 10,

tautomers thereof, geometric isomers thereof and solvates thereof, provided that at least one of R³, R⁴, R⁵ contains an azide group. In embodiments of the first aspect of the invention:

R¹ is Ci-₃ alkyl and R² is H, or R¹ and R² together with the atoms that they are attached to form a 5- to 6-membered heterocyclic ring; R³ represents H, Y(CH₂)_nN₃, or 0C(=0)CH₂N₃;

R⁴ represents Y(CH₂)_nN₃, (CH₂)_mN⁺(R⁷)₃ or aryl, where the aryl group is unsubstituted or substituted by halo, Ci-₃ alkyl or Y(CH₂)_nN₃;

R⁵ represents H, OR⁸ or Y(CH₂)_nN₃;

R⁶ represents H or OR⁸;

R⁷ and R⁸ independently represent at each occurrence Ci-₃ alkyl;

Y represents NR⁹ or O;

R⁹ represents H or Ci-₃ alkyl;

n is a number from 2 to 5; and

m is a number from 2 to 5.

In further embodiments of the first aspect of the invention:

R¹ is CH3 and R² is H, or R¹ and R² together with the atoms that they are attached to form a 6-membered heterocyclic ring;

R³ represents H, Y(CH₂)_nN₃, or 0C(=0)CH₂N₃;

R⁴ represents Y(CH₂)₂N₃, (CH₂)₂N⁺(R⁷)₃ or aryl, where the aryl group is unsubstituted or substituted by CH₃ or Y(CH₂)_nN₃;

R⁵ represents H, OCH₃ or Y(CH₂)_nN₃;

R⁶ represents H or OCH₃; and

Y represents NH or O.

For example, in certain embodiments above, only one of R³, R⁴ and R⁵ may contain an azide group.

Compounds of formula (I) that may be mentioned with respect to the first aspect of the invention as embodiments thereof include:

For example, the compound of formula I may be:

10 In a second aspect of the invention, there is provided a method of detecting and/or quantitating the presence of metabolically active microorganisms in a test sample, the method comprising:

(a) providing a test sample comprising microorganisms;

(b) contacting said microorganisms with a compound of formula I for a period in the dark;

(c) exposing said microorganisms from (b) to light irradiation to activate said compound;

(d) amplifying a target region of DNA of said microorganisms from step (c) by a nucleic acid amplification method; and

(e) comparing the level of amplified product from (d) with that of a control amount of said microorganism.

In embodiments of the second aspect of the invention:

(i) DNA may be isolated from the exposed microorganisms from step (c) prior to amplification in step (d);

(ii) the microorganism may be selected from one or more of the group consisting of bacteria, fungi and yeast;

(iii) the DNA in step (d) may be amplified using a method selected from the group comprising Polymerase chain reaction (PCR), Quantitative polymerase chain reaction (qPCR), Strand displacement amplification (SDA), Helicase-dependent amplification (HDA), Nicking enzyme amplification reaction (NEAR) and loop-mediated isothermal amplification (LAMP);

(iv) the test sample may be selected from the group comprising foodstuff, a biological sample, drinking water, industrial water, environmental water, wastewater, soil and clinical samples.

In a third aspect of the invention, there is provided a microorganism metabolic activity test kit to detect and/or quantitate the presence of metabolically active microorganisms in a test sample, the kit comprising:

(a) a compound of formula I;

(b) amplification primers that target a region of a DNA of the said microorganisms, preferably, wherein at least one of the said primers is structurally and/or chemically modified from its corresponding natural nucleic acid.

In embodiments of the third aspect of the invention: (I) the microorganism may be selected from the group comprising bacterial cells, fungi and yeast cells;

(II) one or more structural and/or chemical modifications may be selected from the group comprising the addition of tags, such as fluorescent tags, radioactive tags, biotin, a 5’ tail, the addition of phosphorothioate (PS) bonds, 2'-0-Methyl modifications and/or phosphoramidite C3 Spacers during synthesis.

Drawings

Figure 1 provides the chemical structure of DyeTox13 Green C2-Azide. A compound of the invention.

Figure 2 depicts bar graphs relating to the ACt of PMA-PCR assays for P. aeruginosa PA01 at various concentrations (10 to 200 mM) for: (A) PMA; (B) EMA; and (C) DyeTox13 Green C2-azide. The ACt = Ct without dye - Ct with dye. The left-hand column at each concentration (when visible) represents the ACt obtained when live cells were used, while the right-hand column at each concentration represents the ACt when dead cells were used. Error bars represent means and standard deviations, which were obtained in three independent replicates.

Figure 3 depicts bar graphs relating to the ACt of PMA-PCR assays for E. faecalis V583 at various concentrations (10 to 200 pM) for: (A) PMA; (B) EMA; and (C) DyeTox13 Green C2- azide. The ACt = Ct without dye - Ct with dye. The left-hand column at each concentration (when visible) represents the ACt obtained when live cells were used, while the right-hand column at each concentration represents the ACt when dead cells were used. Error bars represent means and standard deviations, which were obtained in three independent replicates.

Figure 4 depicts a bar graph relates to the log copies/pL number of live and heat-killed E. faecalis V583 cells in mixtures treated with DyeTox13 Green C2 Azide. The bar graph presents this information as differences in ALog (Log without DyeTox13 Green C2 Azide - Log with DyeTox13 Green C2 Azide) values. The associated table shows the mixing ratios of live and dead cells in each mixture tested. The mean values and error bars were calculated from three independent replicates. Figure 5 depicts epi-fluorescent images obtained by treatment of live and dead cells with PMA, EMA and DyeTox13 Green C2 Azide of (A) P. aeruginosa PA01 and (B) E. faecalis V583. The white scale bars are each 10 pm in length.

Figure 6 depicts epi-fluorescent images obtained by treatment of live and dead cells with PMA+SYTO 9, EMA+SYTO 9 and DyeTox13 Green C2 Azide+PI of (A) P. aeruginosa PA01 and (B) E. faecalis V583. The white scale bars are each 10 pm in length. In each panel, the image of the PMA (left), EMA (middle), and DyeTox13 Green C2 Azide. The upper left image of live cells were stained with PMA, EMA or DyeTox13 Green C2 Azide and the upper right image of live cells were stained with PI. The lower image showed the dead cells were stained with PMA, EMA or DyeTox13 Green C2 Azide and PI, respectively.

Figure 7 A depicts spectrophotometric data obtained by the use of fluorescein diacetate (FDA) in P. aeruginosa PA01 and E. faecalis V583, respectively. The white and grey bars represent live and dead cells respectively, and the error bars represent means and standard deviations obtained in three replicates. Panel B shows the epi-fluorescent images obtained by treatment of P. aeruginosa PA01 and E. faecalis V583 cells with DyeTox13 Green C2-Azide in LB/BHI medium (denoted as“medium”) and PBS buffer, respectively. The insert text shows the mean fluorescence intensity of each the images.

Figure 8 depicts the effect of varying the exposure of gram positive and negative cells to UV light and subsequent analysis of the resulting viable and non-viable cell counts by flow cytometry. Panels A and B show gram negative of E. coli and P. aeruginosa PA01 , respectively. Panels C and D show gram positive of E. faecalis and B. sphaericus, respectively. The line plots denoted by square point values relate to viable cell log numbers, while the line plots denoted by circular point values relate to non-viable cell log numbers.

Figure 9 depicts the effect of varying the exposure of gram positive and negative cells to UV light and subsequent analysis of the resulting viable and non-viable cell counts by PMA and DyeTox13 Green C2-Azide-qPCR. Panels A and B show gram negative of E. coli and P. aeruginosa PA01 , respectively. Panels C and D show gram positive of E. faecalis and B. sphaericus, respectively. PMA and DyeTox13 Green C2-Azide treatment results are shown in ACt (with - w/o Dyes) based on PMA and DyeTox13 Green C2-Azide-qPCR. The white and black bars show the PMA and DyeTox13 Green C2-Azide ACt values, respectively. Error bars represent means and standard deviations, which were obtained in three independent replicates. Figure 10 depicts the effect of varying the exposure of gram positive and negative cells to chlorine and subsequent analysis of the resulting viable and non-viable cell counts by flow cytometry. Panels A and B show gram negative of E. coli and P. aeruginosa PA01 , respectively. Panels C and D show gram positive of E. faecalis and B. sphaericus, respectively. The line plots denoted by square point values relate to viable cell log numbers, while the line plots denoted by circular point values relate to non-viable cell log numbers.

Figure 11 depicts the effect of varying the exposure of gram positive and negative cells to chlorine and subsequent analysis of the resulting viable and non-viable cell counts by PMA and DyeTox13 Green C2-Azide-qPCR. Panels A and B show gram negative of E. coli and P. aeruginosa PA01 , respectively. Panels C and D show gram positive of E. faecalis and B. sphaericus, respectively. PMA and DyeTox13 Green C2-Azide treatment results are shown in ACt (with - w/o Dyes) based on PMA and DyeTox13 Green C2-Azide-qPCR. The white and black bars show the PMA and DyeTox13 Green C2-Azide ACt values, respectively. Error bars represent means and standard deviations, which were obtained in three independent replicates.

Figure 12 depicts the reaction scheme for the synthesis of DyeTox13 Green C2-Azide dye of the current invention.

Description

It has been surprisingly found that the compound DyeTox13 Green C-2 Azide as shown in Fig. 1 and analogues thereof can be used in PCR methods to quantitatively detect only cells with metabolic activity.

Thus, in a first aspect of the invention, there is provided a compound of formula I:

where:

R¹ is C alkyl and R² is H, or R¹ and R² together with the atoms that they are attached to form a 5- to 8-membered heterocyclic ring;

R³ represents H, Y(CH₂)nN₃, or 0C(=0)CH₂N₃;

R⁴ represents Y(CH₂)_nN₃, (CH₂)_mN⁺(R⁷)₃ or aryl, where the aryl group is unsubstituted or substituted by halo, Ci-e alkyl or Y(CH₂)_nN₃;

R⁵ represents H, OR⁸ or Y(CH₂)_nN₃;

R⁶ represents H or OR⁸;

R⁷ and R⁸ independently represent at each occurrence Ci-e alkyl;

Y represents NR⁹ or O;

X represents one or more counter ions to balance the positive charge of the organic molecule; R⁹ represents H or Ci-e alkyl;

n is a number from 2 to 10;

m is a number from 2 to 10,

tautomers thereof, geometric isomers thereof and solvates thereof, provided that at least one of R³, R⁴, R⁵ contains an azide group. It is believed that the compounds of formula I (e.g. DyeToxl 3 Green C-2 Azide) can penetrate cell membranes and so can bind to the dsDNA of both live and dead cells (e.g. see the examples below). In contrast to former dyes, the compounds of formula I are labile when in the presence of a functional cellular metabolism and the azide group of these molecules may be cleaved by a suitable enzyme (e.g. selected from one or more of esterases, oxidoreductases, and other redox enzymes). However, when introduced to a dead cell’s dsDNA, the azide group will not be cleaved due to the lack of metabolic activity, meaning that the azide group can be chemically activated to react upon exposure to light to form covalent bond(s) with the DNA of inactive/dead cells. That is, upon light exposure, the photoactive phenyl azide is converted to a nitrene group that can initiate addition reactions with double bonds, insert into C-H and N-H sites, or undergo subsequent ring expansion with the DNA from inactive cells, meaning that the DNA of dead cells will not undergo PCR amplification due to the presence of these covalent bonds. In contrast, for metabolically active (alive) cells, as the azide will be cleaved before the treated cells are exposed to light, the light exposure does not result in the formation of covalent bonds between the metabolised compound of formula I and the DNA of the live cell, and so PCR amplification will occur (see examples below).

Typically, the sample will be incubated with the compounds of formula I for a period of from 5 to 10 minutes, which is intended to provide sufficient time for the microbial cells in the sample to absorb the compound of formula I and for any live cells to cleave the azide bond. The subsequent light exposure step may then be started at from 5 to 20 minutes following the initiation of incubation with the compound of formula I. The light exposure may occur for any sufficient length of time to ensure reaction of the azide in uncleaved compounds of formula I with the DNA of inactive/dead cells. Examples of a suitable minimum amount of time may be from 5 to 10 minutes of light exposure.

[What is the minimum and maximum amount of time needed to:

(a) cleave the azide for incubation for 5 to 10 min : and

(b) retain the cleaved dye for light exposure for 5 to 20 min in live cells?

I assume that the DNA extraction protocol will flush the dye from the DNA of the live cells, so that it does not affect the PCR amplification. Please confirm. Yes

In addition, please let me know the minimum amount of time that the light needs to be provided to dead cells to cause the reaction of the dye with the DNA. Minimum for about 5 min to 10 min

If you do not know the exact answers, please provide me with your best guesses.]

In embodiments herein, the word“comprising” may be interpreted as requiring the features mentioned, but not limiting the presence of other features. Alternatively, the word“comprising” may also relate to the situation where only the components/features listed are intended to be present (e.g. the word“comprising” may be replaced by the phrases“consists of” or“consists essentially of”). It is explicitly contemplated that both the broader and narrower interpretations can be applied to all aspects and embodiments of the present invention. In other words, the word“comprising” and synonyms thereof may be replaced by the phrase“consisting of” or the phrase“consists essentially of’ or synonyms thereof and vice versa.

References herein (in any aspect or embodiment of the invention) to compounds of formula I includes references to such compounds per se, to tautomers of such compounds, as well as to salts or solvates of such compounds.

Salts of the compound of formula I that may be mentioned include acid addition salts and base addition salts. Such salts may be formed by conventional means, for example by reaction of a free acid or a free base form of a compound of formula I with one or more equivalents of an appropriate acid or base, optionally in a solvent, or in a medium in which the salt is insoluble, followed by removal of said solvent, or said medium, using standard techniques (e.g. in vacuo, by freeze-drying or by filtration). Salts may also be prepared by exchanging a counter-ion of a compound of formula I in the form of a salt with another counter-ion, for example using a suitable ion exchange resin.

Examples of salts include acid addition salts derived from mineral acids and organic acids, and salts derived from metals such as sodium, magnesium, or preferably, potassium and calcium.

Examples of acid addition salts include acid addition salts formed with acetic, 2,2- dichloroacetic, adipic, alginic, aryl sulphonic acids (e.g. benzenesulphonic, naphthalene-2- sulphonic, naphthalene-1 , 5-disulphonic and p-toluenesulphonic), ascorbic (e.g. L-ascorbic), L-aspartic, benzoic, 4-acetamidobenzoic, butanoic, (+) camphoric, camphor-sulphonic, (+)- (1 S)-camphor-10-sulphonic, capric, caproic, caprylic, cinnamic, citric, cyclamic, dodecylsulphuric, ethane-1 , 2-disulphonic, ethanesulphonic, 2-hydroxyethanesulphonic, formic, fumaric, galactaric, gentisic, glucoheptonic, gluconic (e.g. D-gluconic), glucuronic (e.g. D-glucuronic), glutamic (e.g. L-glutamic), a-oxoglutaric, glycolic, hippuric, hydrobromic, hydrochloric, hydriodic, isethionic, lactic (e.g. (+)-L-lactic and (±)-DL-lactic), lactobionic, maleic, malic (e.g. (-)-L-malic), malonic, (±)-DL-mandelic, metaphosphoric, methanesulphonic, 1- hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic, palmitic, pamoic, phosphoric, propionic, L-pyroglutamic, salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulphuric, tannic, tartaric (e.g.(+)-L-tartaric), thiocyanic, undecylenic and valeric acids.

Particular examples of salts are salts derived from mineral acids such as hydrochloric, hydrobromic, hypochloric, phosphoric, metaphosphoric, nitric and sulphuric acids; from organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, arylsulphonic acids; and from metals such as sodium, magnesium, or preferably, potassium and calcium.

As mentioned above, also encompassed by formula I are any solvates of the compounds and their salts. Preferred solvates are solvates formed by the incorporation into the solid state structure (e.g. crystal structure) of the compounds of the invention of molecules of a non-toxic pharmaceutically acceptable solvent (referred to below as the solvating solvent). Examples of such solvents include water, alcohols (such as ethanol, isopropanol and butanol) and dimethylsulphoxide. Solvates can be prepared by recrystallising the compounds of the invention with a solvent or mixture of solvents containing the solvating solvent. Whether or not a solvate has been formed in any given instance can be determined by subjecting crystals of the compound to analysis using well known and standard techniques such as thermogravimetric analysis (TGE), differential scanning calorimetry (DSC) and X-ray crystallography.

The solvates can be stoichiometric or non-stoichiometric solvates. Particularly preferred solvates are hydrates, and examples of hydrates include hemihydrates, monohydrates and di hydrates.

For a more detailed discussion of solvates and the methods used to make and characterise them, see Bryn et ai, Solid-State Chemistry of Drugs, Second Edition, published by SSCI, Inc of West Lafayette, IN, USA, 1999, ISBN 0-967-06710-3.

Compounds of formula I, as well as salts and solvates of such compounds are, for the sake of brevity, hereinafter referred to together as the“compounds of formula I”.

Compounds of formula I may contain double bonds and may thus exist as E ( entgegen ) and Z ( zusammen ) geometric isomers about each individual double bond. All such isomers and mixtures thereof are included within the scope of the invention.

Compounds of formula I may exist as regioisomers and may also exhibit tautomerism. All tautomeric forms and mixtures thereof are included within the scope of the invention.

Compounds of formula I may contain one or more asymmetric carbon atoms and may therefore exhibit optical and/or diastereoisomerism. Diastereoisomers may be separated using conventional techniques, e.g. chromatography or fractional crystallisation. The various stereoisomers may be isolated by separation of a racemic or other mixture of the compounds using conventional, e.g. fractional crystallisation or HPLC, techniques. Alternatively the desired optical isomers may be made by reaction of the appropriate optically active starting materials under conditions which will not cause racemisation or epimerisation (i.e. a‘chiral pool’ method), by reaction of the appropriate starting material with a‘chiral auxiliary’ which can subsequently be removed at a suitable stage, by derivatisation (i.e. a resolution, including a dynamic resolution), for example with a homochiral acid followed by separation of the diastereomeric derivatives by conventional means such as chromatography, or by reaction with an appropriate chiral reagent or chiral catalyst all under conditions known to the skilled person. All stereoisomers and mixtures thereof are included within the scope of the invention.

The term“halo”, when used herein, includes references to fluoro, chloro, bromo and iodo.

Unless otherwise stated, the term“aryl” when used herein includes Ce-u (such as Ce-io) aryl groups. Such groups may be monocyclic, bicyclic or tricyclic and have between 6 and 14 ring carbon atoms, in which at least one ring is aromatic. The point of attachment of aryl groups may be via any atom of the ring system. However, when aryl groups are bicyclic or tricyclic, they are linked to the rest of the molecule via an aromatic ring. Ce-14 aryl groups include phenyl, naphthyl and the like, such as 1 ,2,3,4-tetrahydronaphthyl, indanyl, indenyl and fluorenyl. Embodiments of the invention that may be mentioned include those in which aryl is phenyl.

Unless otherwise stated, the term “alkyl” refers to an unbranched or branched, cyclic, saturated or unsaturated (so forming, for example, an alkenyl or alkynyl)hydrocarbyl radical, which may be substituted or unsubstituted (with, for example, one or more halo atoms). Where the term“alkyl” refers to an acyclic group, it is preferably CM_O alkyl and, more preferably, Ci-e alkyl (such as ethyl, propyl, (e.g. n-propyl or isopropyl), butyl (e.g. branched or unbranched butyl), pentyl or, more preferably, methyl). Where the term“alkyl” is a cyclic group (which may be where the group “cycloalkyl” is specified), it is preferably C_3-12 cycloalkyl and, more preferably, C5-10 (e.g. C5-7) cycloalkyl.

Further embodiments of the invention that may be mentioned include those in which the compound of formula I is isotopically labelled. However, other, particular embodiments of the invention that may be mentioned include those in which the compound of formula I is not isotopically labelled.

The term "isotopically labelled", when used herein includes references to compounds of formula I in which there is a non-natural isotope (or a non-natural distribution of isotopes) at one or more positions in the compound. References herein to "one or more positions in the compound" will be understood by those skilled in the art to refer to one or more of the atoms of the compound of formula I. Thus, the term "isotopically labelled" includes references to compounds of formula I that are isotopically enriched at one or more positions in the compound.

The isotopic labelling or enrichment of the compound of formula I may be with a radioactive or non-radioactive isotope of any of hydrogen, carbon, nitrogen, oxygen, sulfur, fluorine, chlorine, bromine and/or iodine. Particular isotopes that may be mentioned in this respect include ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N, ¹⁵0, ¹⁷0, ¹⁸0, ³⁵S, ¹⁸F, ³⁷CI, ⁷⁷Br, ⁸²Br and ¹²⁵l).

When the compound of formula I is labelled or enriched with a radioactive or nonradioactive isotope, compounds of formula I that may be mentioned include those in which at least one atom in the compound displays an isotopic distribution in which a radioactive or non radioactive isotope of the atom in question is present in levels at least 10% (e.g. from 10% to 5000%, particularly from 50% to 1000% and more particularly from 100% to 500%) above the natural level of that radioactive or non-radioactive isotope.

Compounds of formula I that may be mentioned herein include those in which:

R¹ is C_1-3 alkyl and R² is H, or R¹ and R² together with the atoms that they are attached to form a 5- to 6-membered heterocyclic ring;

R³ represents H, Y(CH₂)_nN₃, or 0C(=0)CH₂N₃;

R⁴ represents Y(CH₂)_nN₃, (CH₂)_mN⁺(R⁷)₃ or aryl, where the aryl group is unsubstituted or substituted by halo, Ci-₃ alkyl or Y(CH₂)_nN₃;

R⁵ represents H, OR⁸ or Y(CH₂)_nN₃;

R⁶ represents H or OR⁸;

R⁷ and R⁸ independently represent at each occurrence Ci-₃ alkyl;

Y represents NR⁹ or O;

R⁹ represents H or Ci-₃ alkyl;

n is a number from 2 to 5; and

m is a number from 2 to 5.

More particularly, compounds of formula I that may be mentioned herein include those in which:

R¹ is CH₃ and R² is H, or R¹ and R² together with the atoms that they are attached to form a 6-membered heterocyclic ring;

R³ represents H, Y(CH₂)_nN₃, or 0C(=0)CH₂N₃; R⁴ represents Y(CH₂)₂N₃, (CH₂)₂N⁺(R⁷)₃ or aryl, where the aryl group is unsubstituted or substituted by CH3 or Y(CH2)_nN3;

R⁵ represents H, OCH3 or Y(CH2)_nN3;

R⁶ represents H or OCH₃; and

Y represents NH or O.

In the above-mentioned embodiments, it may be the case that only one of R³, R⁴ and R⁵ contains an azide group.

Compounds of formula I that may be mentioned herein include those where the compound of formula I is selected from the list:

For example, the compound of formula I may be DyeTox13 Green C-2 Azide:

As will be appreciated, the compounds disclosed herein may be particularly suited to the detection and/or quantification of metabolically active microorganisms in a test sample obtained from a suitable source. Thus, there is provided a method of detecting and/or quantitating the presence of metabolically active microorganisms in a test sample, the method comprising:

(a) providing a test sample comprising microorganisms;

(b) contacting said microorganisms with a compound of formula I as described above for a period in the dark;

(c) exposing said microorganisms from (b) to light irradiation to activate said compound;

(d) amplifying a target region of DNA of said microorganisms from step (c) by a nucleic acid amplification method; and

(e) comparing the level of amplified product from (d) with that of a control amount of said microorganism. The microorganism to be tested may be any suitable organism, such as a bacteria, a fungus and a yeast or any combination thereof. It will be appreciated that the microorganism(s) may be either Gram positive and/or Gram negative. For example, in certain embodiments, the microorganism(s) may be Gram negative, while in other embodiments the microorganism(s) may be Gram positive, while in yet further embodiments the microorganisms may include both Gram negative and Gram positive microorganism(s).

As will be appreciated, the DNA amplification in step (d) above may be conducted by any suitable means, such as one or more of: Polymerase chain reaction (PCR), Quantitative polymerase chain reaction (qPCR), Strand displacement amplification (SDA), Helicase- dependent amplification (HDA), Nicking enzyme amplification reaction (NEAR), and loop- mediated isothermal amplification (LAMP).

As will be understood, any suitable target region of one or more genes of said microorganism may be selected for DNA amplification. A non-limiting example is the GYRB gene of P. aerigunosa, a target region of which may be amplified using oligonucleotide primers, such as those set forth in SEQ ID No: 1 and SEQ ID No: 2. Another non-limiting example is the 165RRNA gene of E. faecalis, a target region of which may be amplified using oligonucleotide primers, such as those set forth in SEQ ID No: 3 and SEQ ID No: 4.

It will be appreciated that the DNA to be subjected to amplification in the above method may be isolated from the microorganisms exposed to the compound of formula I in step (c) above before the DNA amplification of step (d).

As will be appreciated, the method described herein may be used with any suitable sample. Such samples include, but are not limited to, foodstuff, a biological sample, drinking water, industrial water, environmental water, wastewater, soil and clinical samples. In particular embodiments of the invention that may be mentioned herein, the sample may be blood or urine from a subject.

The examples section below provides full details of how a compound of formula I may be used in combination with a DNA amplification method to quantify the amount of live/metabolically active cells remaining in a sample. As shown in the examples section below, this methodology may be particularly useful in determining the number of metabolically active cells remaining in a sample following disinfection treatments with techniques that do not necessarily destroy the cell wall of a microorganism (e.g. UV treatment) and which may result in bacteria entering a viable but nonculturable state. Additionally or alternatively, the methods disclosed herein may be useful in the routine monitoring of metabolically active cells in a sample, such as an environmental water sample. In order to conveniently conduct the method described above, there may also be provided a microorganism metabolic activity test kit to detect and/or quantitate the presence of metabolically active microorganisms in a test sample, the kit comprising:

(a) a compound of formula I as described above; and

(b) amplification primers that target a region of a DNA of the said microorganisms, preferably, wherein at least one of the said primers is structurally and/or chemically modified from its corresponding natural nucleic acid.

As will be appreciated, the amplification primers for the microorganisms may be selected to selectively bind to and amplify a gene or genes of a species of microorganism selected from the group comprising bacteria, fungus and yeast.

Non-limiting examples of suitable amplification primers include those set forth in may be amplified using oligonucleotide primers, such as those set forth in SEQ ID No: 1 and SEQ ID No: 2, directed to the GYRB gene of P. aerigunosa and/or those set forth in SEQ ID No: 3 and SEQ ID No: 4, directed to the 165RRNA gene of E. faecalis.

As will be appreciated, one or more structural and/or chemical modifications may be applied to the amplification primers to, for example, improve their stability or be used for detection. These modification may include, but are not limited to, the addition of tags, such as fluorescent tags, radioactive tags, biotin, a 5’ tail, the addition of phosphorothioate (PS) bonds, 2 -0- Methyl modifications and/or phosphoramidite C3 Spacers during synthesis.

Further details of the invention are provided by the following non-limiting examples.

Examples

Material and Methods - Examples 1 to 3

In Examples 1 to 3, we evaluated the efficacy of DyeTox13 Green C-2 Azide dye combined with quantitative PCR (qPCR) using the gram-negative ( Pseudomonas aeruginosa PA01) and gram-positive ( Enterococcus faecalis v 583) bacteria. The DyeTox13 Green C2-Aizde dye was compared to existing molecular assays such as EMA-qPCR and PMA-qPCR to test its capability in discriminating metabolically active (viable) cells from metabolically inactive or dead cells.

Strains and culture conditions Pseudomonas aeruginosa PA01 and Enterococcus faecalis v583 were used as model microorganisms for gram-negative and positive bacteria, respectively. A single colony from the overnight culture of Luria broth (Sigma-Aldrich, USA) agar plate of P. aeruginosa PA01 was transferred to 5 ml_ of Luria Bertani (LB) broth and it was grown to a mid-exponential phase (37°C and 200 rpm). E. faecalis v583 was aerobically cultured for 16 hours at 37°C in brain heart infusion (BHI) broth (Sigma-Aldrich, USA). The final cell concentrations of P. aeruginosa PA01 and E. faecalis v583 were adjusted at Oϋboo equal to 1.0 and OD495 equal to 1.0, respectively.

Preparation of heat-killed cells

Each aliquot suspension (500 pL) of P. aeruginosa PA01 and E. faecalis v583 was adjusted to an optical density of 1.0 (approximately from 10⁵ to 10⁶ CFU/mL) by being diluted with fresh BHI and LB broth. Prior to the EMA, PMA, and DyeTox13 Green C-2 Azide treatment, suspensions in 500 pL aliquots (approximately from 10⁵ to 10⁶ CFU/mL) were heated in a water bath for 15 minutes at 99°C (for P. aeruginosa PA01) or 30 minutes at 95°C (for E. faecalis v583). After the treatments, the cells were immediately placed on ice. The loss of viability was determined in triplicate by spread plating 100 pL of heat-killed cells on BHI and LB plates and incubated for 48 hours at 37°C.

Example 1

EMA, PMA, and DyeToxl 3 Green C-2 Azide treatment

EMA (Biotium, Hayward, CA, USA) and PMA (Biotium, Hayward, CA, USA) were dissolved in 20% dimethyl sulfoxide (DMSO, Sigma-Aldrich, Singapore; 20% by volume DMSO in 80% H2O) to obtain a stock solution of 20 mM. The DyeTox13 Green C-2 Azide was synthesised by Setareh Biotech (Eugene, OR, USA), in accordance to the scheme depicted in Fig. 12, and was dissolved in 20% DMSO (by volume in 80 vol% H2O). Each dye solution was stored at - 20°C in the dark prior to use. Each dye solution was added to a total volume of 500 pL of viable and heat-killed (dead) cells to obtain a final concentration of 10, 50, 100, and 200 pM. Following the addition of the dyes, samples were incubated for 10 minutes in the dark at room temperature. The samples were mixed occasionally. After incubation, sample tubes were exposed to a PMA-Lite™ LED Photolysis device (Biotium, Hayward, CA, USA) for 15 minutes. To determine DyeTox13 Green C-2 Azide’s ability to distinguish between viable and heat- killed cells within a mixture, E. faecalis v583 cells (95°C for 30 min) were mixed with the viable cells in defined ratios with relative proportions of 100%, 50%, 10%, 1 %, 0.1 %, and 0% of the total cell concentration of 10⁵-10⁶ CFU. Each mixture was treated with 50 mM of DyeTox13 Green C-2 Azide. After 10 minutes of incubation in the dark at room temperature (can be mixed once, or with continuous mixing), the mixtures were exposed to light for 15 minutes as described above.

DNA extraction and Real-time PCR conditions

Genomic DNA was extracted from the 500 pl_ of EMA-, PMA- or DyeTox13 Green C-2 Azide- treated/untreated samples using a GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific, USA) according to the manufacturer’s protocol. The amount of DNA present was determined using the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). Quantitative PCR was performed using an ABI Step-One-Plus Real- Time PCR system (Applied Biosystems, CA, USA). Quantitative PCR primer sets were selected as described in Table 1.

Table 1. Target genes and primers used in this study

For quantitative PCR, 2 mI_ of genomic DNA (containing from 50 ng to 200 ng DNA) was added to a total volume of 20 mI_ mixture, containing 2 x PowerUp SYBR Green PCR Master Mix (Applied Biosystems, CA, USA) 10 mI_, 300 nM of forward primer 0.6 mI_, 300 nM reverse primer 0.6 mI_ and 6.8 mI_ of PCR-grade sterile water. The qPCR assays were conducted under the following conditions: holding at 50°C for 2 minutes and 95°C for 2 minutes, followed by 40 cycles of 95°C for 15 seconds, 60°C for 15 seconds, and 72°C for 1 minute. Melting-curve analysis was performed to verify the amplification specificity at 95°C for 15 seconds, 60°C for 1 minute, and 95°C for 15 seconds. A standard curve for the qPCR assay was generated by serially diluting the plasmids containing each target gene (10² to 10⁹ copies per reaction). Each amplified gene fragment from the three strains was purified using a GeneJET PCR Purification Kit (Thermo Fisher Scientific, USA) and ligated into the pGEM-T Easy Vector respectively, following the manufacturer's instructions (Promega, USA). The pGEM-T Easy Vector containing the inserts was transformed on JM 109 competent cells, and the transformants were selected on LB plates containing ampicillin (100 pg/mL), 100 mM of IPTG (isopropyl^-Dthiogalactopyranoside), and 50 mg/mL of X-Gal (5-bromo-4-chloro-3- indolyl^-D-galactopyranoside). The cloned vectors were purified using the QIAprep Spin Miniprep Kit (Qiagen, Valencia, CA, USA) and the concentration of the vector DNA was quantified with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). Standard curves were prepared using serial 10-fold dilutions of the pGEM-T Easy Vector containing each target gene. To evaluate the effect of each dye on treated and untreated samples, the AC_t and ALog were calculated by subtracting the C_t values (or the numbers of the log genome copies) of the dye treated samples from the C_t values of the untreated samples.

Results: Effect of EMA, PMA, and DyeTox 13 Green C-2 Azide on Gram-negative organisms

DyeTox13 Green C-2 Azide dye as compared to EMA and PMA against Gram-negative organisms.

Viable and heat-killed P. aeruginosa PA01 cells were exposed to different concentrations of EMA, PMA, and DyeTox13 Green C-2 Azide (10, 50, 100, and 200 mM) using the procedure set out above. The results of EMA, PMA, and DyeTox13 Green C-2 Azide-qPCR were expressed as the changes in the average AC_t, calculated by subtracting the C_t value of the dye treated samples from the C_t value of the non-treated samples. The PMA treatment of the viable P. aeruginosa PA01 cells showed almost no difference between the dye-treated and non- treated samples (bars on the left hand side of Figs. 2A and 2B), while slight AC_t changes were observed in the EMA treatment. On the other hand, both EMA and PMA treatments suppressed the qPCR amplification of the heat-killed cells (right-hand bars in Figs. 2A and 2B). The effect of the EMA and PMA concentrations on PCR amplifications of P. aeruginosa cells showed trends similar to those seen in a previous study, where P. aeruginosa cells were treated with heat (Reyneke, B., etal 2017 Applied Microbiology and Biotechnology. 101 , 7371- 7383). Similar to the EMA and PMA-qPCR analysis, no differences were observed when DyeTox13 Green C-2 Azide was used to treat viable cells (left hand bars in Fig. 2C). However, the AC_t difference in the heat-killed cells was observed in the range of 5 to 12 (right hand bars in Fig. 2C). These results indicated that dead cells of P. aeruginosa PA01 cells were effectively suppressed when treated with the DyeTox13 Green C-2 Azide. To improve the efficiency of the DyeTox13 Green C-2 Azide-qPCR assay, we also investigated the effect of extending incubation times (30 and 60 minutes) of the DyeTox13 Green C-2 Azide dye, while keeping the dye concentration (100 mM) and light exposure time (15 min) constant. The AC_t values showed no difference, regardless of incubation times, to the results shown in Fig. 2C (data not shown).

Results: Effect of EM A, PM A, and DyeTox13 Green C-2 Azide on Gram-positive organisms

E. faecalis v583 cells were treated with the DyeTox13 Green C-2 azide to determine if the newly developed dye could discriminate metabolically active (viable) cells from metabolically inactive or dead cells of gram-positive bacteria.

As described above, all experiments were performed in the different concentrations of EMA, PMA, and DyeTox13 Green C-2 Azide dyes (10, 50, 100, and 200 pM). The effects of the EMA, PMA, and DyeTox13 Green C-2 Azide-qPCR assays on viable and heat-killed E. faecalis v583 cells are presented in Fig. 3. PMA treatment caused the heat-killed E. faecalis v583 to exhibit a considerable difference (9 to 19) in AC_t values when the PMA concentrations were increased (right-hand bars in Fig. 3A). The viable cells exhibited no difference in the C_t values of the PMA-treated and PMA-untreated cells (left-hand bars in Fig. 3A). When treated by EMA, on the other hand, AC_t values in both viable and heat-killed cells increased gradually with increasing concentrations of EMA (Fig. 3B). It has also been reported that EMA treatment can penetrate viable cells (0.10 to 0.76 log decrease) of E. faecalis (Reyneke, et al 2017 Applied Microbiology and Biotechnology. 101 , 7371-7383). These results indicated that EMA penetrated not only viable cells but also dead cells. Several reports have shown that EMA could penetrate the viable-cell membranes of some bacteria species, leading to an underestimation of viable cell numbers (e.g. see: Cawthorn, D.M., and Witthuhn, R.C., 2008. J Appl Microbiol. 105, 1178-1185; Kobayashi, H., et a/ 2009 Lett Appl Microbiol. 48, 633-638; and Nocker, A., et al 2006 J Microbiol Methods. 67, 310-320). PMA is more effective in differentiating between viable and dead cells due to its ability to better penetrate the compromised membranes of dead cells as compared to EMA. However, the effectiveness of PMA and EMA varied depending on their concentration, the bacteria species, incubation time, and light exposure time (Fittipaldi, M., et al 2012. J Microbiol Methods. 91 , 276-289; Flekna, G., et a/2007 Res Microbiol. 158, 405-412; Kobayashi, H., et a/ 2009 J Orthop Res. 27, 1243- 1247; and Wang, L, et al 2009 J Appl Microbiol. 107, 1719-1728). In contrast, exposing heat-killed E. faecalis v583 cells to DyeTox13 Green C-2 Azide treatment resulted in an increase of AC_t values from 11 to 17 (right-hand bars in Fig. 3C). Compared with EMA, DyeTox13 Green C-2 Azide-qPCR dye more effectively discriminated between the viable and dead cells of the organism because it only affected the dead cell signals. DyeTox13 Green C-2 Azide-qPCR also showed that even in low concentrations (10 mM) it was better able to discriminate between viable and dead cells than the PMA-qPCR assay was. Since the mechanism of action (PMA vs. DyeT ox13 Green C-2 Azide) was different, we could not directly compare the efficacy of DyeTox13 Green C-2 Azide with that of the PMA, but the DyeTox13 Green C-2 Azide method showed good results for assessing the viability of E. faecalis v583. Also, DyeTox13 Green C-2 Azide-qPCR more effectively suppressed the PCR amplification of the dead cells of E. faecalis v583 than the dead cells of P. aeruginosa PA01.

To test DyeTox13 Green C-2 Azide dye’s ability to quantify viable cells in the presence of dead cells, DyeTox13 Green C-2 Azide-qPCR assay was applied to a mixture of viable and heat- killed E. faecalis v 583 cells, as shown in Fig.4. Each mixture was subjected to qPCR analysis after being treated with final concentrations of 50 pM DyeT ox13 Green C-2 Azide. The number of gene copies without the DyeTox13 Green C-2 Azide treatment did not show any difference between the mixture of viable cells and heat-killed cells (data not shown). However, after the DyeTox13 Green C-2 Azide treatment, the copy numbers of the mixtures gradually increased in ALogio values with increasing concentration of heat-killed cells (Fig. 4). These results demonstrated that the DyeTox13 Green C-2 Azide treatment successfully quantified viable cells in the mixture of viable and heat-killed E. faecalis v583 cells.

Example 2

Fluorescence microscopy

In order to evaluate the membrane permeability of the viable and dead P. aeruginosa PA01 and E. faecalis v 583 cells, each dye (PMA, EMA, propidium iodide (PI), DyeTox13 Green C- 2 Azide, and SYTO 9) was added to a 500 pL sample of viable and heat-killed bacteria cells. The bacterial cells were incubated for 10 minutes in the dark at room temperature. Samples were analyzed using the Nikon Eclipse 90i epi-fluorescence microscope equipped with a 100x oil immersion objective and a CCD camera (Nikon Corp., Tokyo, Japan). The fluorescence signals of PMA, EMA, DyeTox13 Green C-2 Azide, and SYTO 9 were detected using a FITC (excitation/emission = 494/518 nm) and PI (excitation/emission = 589/615 nm) filter. Image analysis was performed using the NIS-Elements Advanced Research software under fluorescent microscopy (Eclipse 90i, NIS-Element D, Nikon). Results

The usefulness of DyeTox13 Green C-2 Azide in metabolic activity assay was compared with EM A and PMA under epi-fluorescence microscopy, as described previously in Nocker, A., et al 2006 J Microbiol Methods. 67, 310-320. When viable P. aeruginosa PA01 and E. faecalis v583 cells were stained with 50 mM PMA, viable cells did not exhibit fluorescence signals. However, dead cells showed the red fluorescence signals caused by PMA (Fig. 5, left). In contrast, EMA exhibited red fluorescence signals both with viable and dead P. aeruginosa PA01 and E. faecalis v583 cells, although viable E. faecalis v583 cells showed lower fluorescence signals than dead cells did (Fig. 5, middle). Moreover, DyeTox13 Green C-2 Azide (green fluorescence) exhibited in both viable and dead P. aeruginosa PA01 and E. faecalis v583 cells (Fig. 5, right). The microscopic results proved that PMA penetrated only dead cells with compromised membranes, whereas EMA could penetrate both viable and dead P. aeruginosa PA01 and E. faecalis v 583 cells.

DyeTox13 Green C-2 Azide stained cells exhibited green fluorescence signals in both viable and dead cells. We further tested the efficacy of DyeTox13 Green C-2 Azide in discriminating between metabolically active and metabolically inactive or dead cells using a combination of the dyes such as PMA/SYT09, EMA/SYT09, and DyeTox13 Green C-2 Azide/PI, following the procedure used by Nocker, A., et al 2006 J Microbiol Methods. 67, 310-320. When stained with a PMA/SYT09 and DyeTox13 Green C2-azide/PI, viable cells exhibited green (SYT09 or DyeTox13 Green C2-azide) fluorescence signals but not red (PMA or PI), while the dead cells showed both green (SYT09 or DyeTox13 Green C2-azide) and red (PMA or PI) fluorescence signals (Fig. 6, left and right). The same trend was observed in EMA/SYT09 (Fig. 6, middle) when stained with EMA alone (Fig. 6, middle). The results of this study proved that DyeTox13 Green C-2 Azide could bind to DNA from both live and inactive cells, as with the SYTO™ 9 Green stain. In addition, these results implied that DyeTox13 Green C-2 Azide can selectively detect the viable and metabolically active cells as seen in the qPCR results above.

Example 3

Microbial activity test

To stain the cells with fluorescein diacetate (FDA), we followed the protocols described by Schnurer and Rosswall (1982 Applied and Environmental Microbiology 43, 1256-1261). FDA (Thermo Fisher Scientific, USA) was dissolved in acetone to obtain a 2 mg/ml_ stock solution, which was stored at -20°C. The viable and heat-killed P. aeruginosa PA01 and E. faecalis v583 cells were prepared as described above. After they were grown, cell concentrations were adjusted to 1.0 for optical density with 50 ml_ of either fresh LB or BHI medium. FDA was added to aliquots of bacteria culture to a final concentration of 200 pg/mL. The bacterial suspensions were incubated on a rotary shaker (with 100 rpm) at 24°C for 60 minutes. After incubation, acetone (50% v/v) was added to terminate the FDA hydrolysis. Cell debris was eliminated by centrifugation at 6,000 rpm for 5 minutes and the supernatants were then filtered. The fluorescein concentration was measured at 490 nm using a spectrophotometer (Hitachi U-2800 UV-Vis Spectrophotometer).

To test for the effect of DyeTox13 Green C-2 Azide dye use on microbial activity, metabolically active and inactive cells were prepared under fresh medium (LB or BHI) and PBS buffer (Vivantis Technologies, Malaysia). DyeTox13 Green C-2 Azide dye was added to fresh medium (active cells) and PBS buffer (inactive cells) of bacteria culture to a final concentration of 50 pM and incubated for 60 minutes in the dark at 37°C. The fluorescence images were collected with a Nikon DS-Ri1 camera in a Nikon Eclipse 90i epi-fluorescence microscope (Nikon Corp., Tokyo, Japan). The intensity of fluorescence was further analyzed using image processing software ImageJ (NIH, USA).

Results

We investigated how DyeTox13 Green C-2 Azide dye affected the metabolic activity of P. aeruginosa PA01 and E. faecalis v583 cells. Fluorescein diacetate (FDA) has widely been used for studying the microbial activity of soil and bacteria (Adam, G., and Duncan, H., 2001. Soil Biology and Biochemistry. 33, 943-951 ; and Schumacher, T.E., et al 2015 Environmental Science and Pollution Research. 22, 4759-4762). The FDA assay is based on the activity of FDA hydrolysis by a number of different enzymes (e.g. esterases, lipases and proteases - see Guilbault, G.G. and Kramer, D.N., 1964 Analytical Chemistry 36, 409-412; and Rotman, B. and Papermaster, B.W., 1966 Proc Natl Acad Sci USA 55, 134-141). The metabolic activity can be evaluated by fluorescence microscopy or spectrophotometry. Since both FDA and DyeTox13 Green C-2 Azide dyes have similar absorption spectra, and FDA cannot stain all organisms (Dorsey, J., et al 1989 Cytometry 10, 622-628), microbial activity were measured separately, using either the FDA or DyeTox13 Green C-2 Azide dye.

We first assessed microbial activity of cells by vital staining with FDA. Results show that the amount of fluorescein increased by 0.23 (Absorbance at 490 nm, A₄₉₀) for P. aeruginosa PA01 and 0.83 (A₄₉₀) for E. faecalis v583 in viable cells (Fig. 7A, white bars). In contrast, the heat- killed cells showed low levels of fluorescein when compared with viable cells (Fig. 7A, grey bars). The difference of the two organisms’ amount of fluorescein was caused partly by different cellular esterase activity. Also, the amount of fluorescein depends on several parameters including: FDA concentration, incubation time, and the number of cells (Saruyama, N. et a/ 2013. Anal Biochem. 441 , 58-62).

The effect of DyeTox13 Green C-2 Azide dye use on microbial activity was subsequently evaluated by using metabolically active and inactive cells. We analyzed cells’ fluorescence intensity using ImageJ software. When viable P. aeruginosa PA01 and E. faecalis v583 cells grown in medium and buffer were treated with 50 mM of DyeTox13 Green C-2 Azide dye, the mean of the fluorescence intensity (arbitrary units) was 27 ± 2 (standard deviation is included as ±) (medium) to 14 ± 7 (buffer) for P. aeruginosa PA01 , and 61 ± 18 (medium) to 29 ± 6 (buffer) for E. faecalis v583 (Fig. 7B). The statistical analysis showed significant differences between the samples that were grown in medium and those that were buffer (P < 0.05). These results indicate that metabolically active cells (medium) showed strong fluorescence intensity, while metabolically inactive cells (buffer) exhibited decreased fluorescence signal intensity. Although the metabolic activity test was simple, it confirmed the metabolic activity in different types of growth conditions.

Materials and Methods - Examples 4-5 and Reference Examples 1-2

General Procedure 1

Bacterial strains and culture conditions

One single colony of E. coli, P. aeruginosa, E. faecalis, and B. sphaericus from Luria-bertani (LB; Sigma-Aldrich, USA) or brain heart infusion (BHI) agar (Sigma-Aldrich, USA) plates were inoculated into 100 mL LB broth and BHI broth and incubated at 37°C with shaking at 200 rpm for 16 h. The cells were transferred to 100 mL of fresh LB and BHI broth in 250 ml flasks with its final concentrations adjusted to be approximately 10⁷ CFU/mL. Colony-forming unit (CFU) counts of culturable bacteria were performed by spreading of serially diluted samples on LB and BHI agar plates.

General Procedure 2

Exposure to UV and Chlorine

The concentrations of NaOCI used were 10, 30 and 50mg/L. UV exposure test was conducted in 1300 Series Class II, Type A2 Biological Safety Cabinet (BSC) equipped with a timed UV system (T. Scientific, Cabinets, & Benches, n.d.). Likewise, triplicates of 10 mL aliquot of each cell suspension were prepared in petri plates for exposure periods of 30 and 60 mins in the BSC. General Procedure 3

Determination of viable and culturable cell counts

Samples were stained before and after each disinfection process using LIVE/DEAD BacLight bacterial viability kit (L7012 LIVE/DEAD™ BacLight™ Bacterial Viability Kit, n.d.) for flow cytometry. The dyes used were 0.167 mM of SYT09 and 1 mM of propidium iodide (PI), giving green and red fluorescence respectively. Cells were suspended in PBS prior to staining, and left in the dark for 15 mins after staining to allow the PI dye to penetrate cells with disrupted membrane integrity. Analyses were performed with CytoFLEX (Beckman Coulter, Singapore) equipped with an Argon Ion Laser tuned to an excitation wavelength of 488 nm. For cell counting purpose, FluoSpheres™ Polystyrene Microspheres, 1.0 pm were obtained from Thermo Fisher Scientific (Singapore) and added into each sample before being processed by the flow cytometer. The number of cells per microliter was calculated based on the following formula:

Number of bacterial cells per mL = (bacteria events)/(beads events) * bead numbers /mL * dilution factor

Multi-parametric analyses were performed on both scattering signals (FSC and SSC), FL1 (FITC to detect green fluorescence) and FL2 (PE to detect red signal). The data were then acquired through CytExpert software provided by Beckman Coulter, and used to determine the quantity of viable and non-viable cell count in each sample exposed to different NaOCI concentration or UV exposure time.

General Procedure 4

Culturable cell counting

The culturability of bacteria was investigated by growing E. coli and P. aeruginosa on LB agar plates, and E. faecalis and B. sphaericus on BHI agar plates. Cells were serially diluted in 1x phosphate-saline buffer (PBS, 137 mM NaCI, 2.7 mM KCI and 10 mM Phosphate Buffer, pH 7.4) (Vivantis Technologies, Malaysia) before spreading 100 m\- of inoculum on triplicate plates for each set of dilution. CFU count was conducted after 24 hours of incubation at 37°C.

General Procedure 5

PMA and DyeTox13 Green C-2 Azide treatment PMA (Biotium, Hayward, CA, USA) and DyeTox13 Green C-2 Azide (Eugene, OR, USA) were dissolved in 20% dimethyl sulfoxide (DMSO, Sigma-Aldrich, Singapore) in order to obtain a 20 mM stock solution (Lee and Bae 2017, 2018). Each 500 pL of cell suspensions was treated with PMA or DyeTox13 Green C-2 Azide to obtain final concentrations of 50 pM. After addition of PMA or DyeTox13 Green C-2 Azide, the cell suspensions were mixed well by vortexing and incubated in the dark at room temperature for 10 min. The samples were exposed to intense visible light for 15 min using the PMA-Lite™ LED Photolysis device (a long-lasting LED Lights with 465-475 nm emission for PMA™ activation; Biotium, Inc.). The PMA or DyeTox13 Green C-2 Azide-treated samples were then pelleted by centrifugation (5,000 x g for 10 min) and DNA extracted from the pallet.

General Procedure 6

DNA extraction and Real-time PCR assays

DNA was extracted from E. coli, P. aeruginosa, E. faecalis, and B. sphaericus using a DNeasy Blood and Tissue Kit (Giagen, Valencia, CA, USA) according to the manufacturer’s protocol. Purified DNA concentrations of the samples were quantified using a Cubit 3.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, United States). Real-time PCR was performed in a volume of 20 pL containing 10 pL PowerUp SYBR Green PCR Master Mix (2x) (Applied Biosystems, CA, USA), 2 pL of gDNA, 0.6 pL of forward primer and reverse primer (300 nM) and 6.8 pL of nuclease-free water (IDT). The qPCR reaction conditions were as follows: 50°C for 2 min and 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec, 60°C for 1 min in a StepOne qPCR instrument (Applied Biosystems). All primers were synthesized and purified from Integrated DNA Technologies (IDT) (Table 1). A standard curve was prepared by serial diluted of pGEM-T Easy vector (10⁹-10² copies/pL) containing each target the uidA, gyrB, and 16s rRNA genes as previously study (Ahmad et al. 2017, Lee and Bae 2018).

To evaluate the effect of PMA and DyeTox13 Green C-2 Azide on treated and untreated samples, the AC_t was calculated by subtracting the C_t values of the dye treated samples from the C_t values of the untreated samples. The error bar in this study’s figures indicates the standard deviations from three independent replicates.

Reference Example 1

Monitoring of UV treatment in gram-negative and gram-positive by viability and culturability For E. coli, P. aeruginosa, E. faecalis and B. sphaericus cells exposed to 30 and 60 mins of UV (General Procedure 2), the flow cytometry data revealed an almost constant viable cell count of 10⁷ cells/mL (General Procedure 3). However, the plate counts (General Procedure 4) demonstrated a stark contrast in culturability of all four types of cells (see Fig. 8).

At 30 mins UV exposure, culturable cell counts of E. coli, E. faecalis, and B. sphaericus declined to almost zero. Likewise, for 60 mins UV exposure time, culturable cell counts for these three cell types were zero. However, P. aeruginosa showed a more gradual decrease in culturability. Cell count was 10² cells/mL at 30 mins UV exposure and 10¹ cells/mL at 60 mins exposure. Given that P. aeruginosa culturability did not diminish to zero even after 60 mins of UV exposure, it has shown a greater resistance to UV treatment as compared to E. coli, E. faecalis, and B. sphaericus.

Studies have shown that UV induces thymine dimers in DNA within cells, without injuring the cell membrane. PI stains membrane-permeabilized cells, hence an intact membrane regardless of whether the cellular DNA has undergone dimerization would null the feasibility of using flow cytometry to distinguish live and dead cells in the samples. Although the decrease in plate counts indicated a significant reduction in culturability of all cell types, the flow cytometry data was not able to complement the plate counts to conclude the presence of VBNC cells because it provides an inaccurate representation of cell viability.

Example 4

Monitoring of UV treatment in gram-negative and gram-positive organisms by PMA and DyeTox13 Green C-2 Azide-qPCR analysis

DyeTox13 Green C-2 Azide was investigated for its ability to quantitatively determine the presence of VBNC after UV disinfection compared to PMA. Total four bacteria cells (E. coli, P. aeruginosa, E. faecalis, and B. sphaericus) were first exposed with UV to evaluate UV disinfection efficiency (General Procedure 2). The results of PMA and DyeTox13 Green C-2 Azide-qPCR (General Procedure 6) are shown in Fig. 9 as the change in the average AC_t calculated by subtracting the C_t value of the dye non-treated samples from the C_t value of the treated samples.

All experiments were stained with 50 mM PMA and DyeTox13 Green C-2 Azide according to General Procedure 5. The PMA treatment of the two gram-negative of E. coli and P. aeruginosa cells showed almost no difference between the dye-treated and non-treated samples, although slight fluctuations were observed during the UV exposure (Figs. 9A and 9B, white bars). Furthermore, the DyeTox13 Green C-2 Azide-qPCR results showed a similar tendency to the PMA-qPCR (Figs. 9A and 9B, grey bars). Unlike gram-negative bacteria, DyeTox13 Green C-2 Azide treatment caused the gram-positive of E. faecalis and B. sphaericus to exhibit a considerable difference (7 to 10) in AC_t values after being treated for 60 min (Fig. 9C and 9D, grey bars). Compared to B. sphaericus, E. faecalis was more effectively suppressed by the PCR amplification of nonactive cells (Figs. 9C and 9D, grey bars). On the other hand, PMA-qPCR assay exhibited no differences in AC_t regardless of UV exposure times (Figs. 9C and 9D, white bars). Also, DyeTox13 Green C-2 Azide-qPCR more effectively suppressed the PCR amplification of the nonactive cells of gram-positive bacteria. These results show that DyeTox13 Green C-2 Azide dye-qPCR can be used effectively to distinguish between active and nonactive gram-positive bacteria.

In addition, these results also suggest that the membrane-based viability techniques of PMA- qPCR might be incapable of discriminating VBNC cells from UV inactivated bacteria because treatment with UV does not cause direct cell membrane damage. Many bacterial species can enter a distinct state commonly called the “viable but nonculturable” (VBNC) state as a strategy to survive under unfavorable environmental conditions, such as UV disinfection, osmotic pressures, chemical disinfection, starvation, and extreme temperature. In the VBNC state, the bacteria still remain viable and maintain metabolic activity, VBNC cells pose a public health risk. Therefore, DNA-intercalating dyes which discriminate between viable and non- viable cells based on membrane integrity as a viability criterion (rather than the physiological state of the cells) are generally unable to effectively discriminate under UV disinfection conditions. As such, coupling DyeTox13 Green C-2 Azide with qPCR appears to be a promising method to detect VBNC cells in such environmental conditions.

Reference Example 2

Monitoring of chlorine treatment in gram-negative and gram-positive by viability and culturability

For E. coli, P. aeruginosa, E. faecalis and B. sphaericus cells were exposed to various concentrations of NaOCI using General Procedure 2, and the flow cytometry data was collected using General Procedure 3. These results are presented in Fig. 10A-D.

For viability studies on NaOCI disinfection, a decrease in the viability of E. coli (Fig. 10A), P. aeruginosa (Fig. 10B), and E. faecalis (Fig. 10C) from the initial 10⁷ to 10⁵ cells/mL was observed during exposure to 10 mg/L NaOCI. However, there was no further decrease in viability in the samples exposed to 30 and 50 mg/L NaOCI. The viability of B. sphaericus (Fig. 10D) decreased more gradually as NaOCI concentration increased. Eventually, a final cell count of 10⁵ cells/mL was reached when B. sphaericus was exposed to 50 mg/L NaOCI. The agar plates yielded lower viable counts for all four types of cells. Culturable cell counts for E. coli decreased from initial 10⁶ cells/mL almost zero in the presence of 10, 30, and 50 mg/L NaOCI; culturable cell counts for P. aeruginosa decreased from 10⁷ to 10² cells/mL in the presence of 10 mg/L NaOCI, and almost zero when exposed to 30 and 50 mg/L NaOCI; culturable cell counts for E. faecalis decreased from 10⁷ to 10¹ cells/mL in the presence of 10 mg/L NaOCI, and almost zero when exposed to 30 and 50 mg/L NaOCI; culturable cell counts of B. sphaericus decreased from initial 10⁷ cells/mL almost zero in the presence of 10, 30, and 50 mg/L NaOCI.

Chlorine treatment has proven to oxidize cellular components such as outer membrane and periplasmic proteins of bacterial cells (Gray, Wholey, & Jakob, 2014). ^~OCI inactivates operative proteins concentrated in the plasma membrane, while HOCI is able to further diffuse through the lipid bilayer of the plasma membrane and oxidize intracellular components, a much severe impact on the cells. These oxidizing actions eventually lead to compromised cell membrane integrity, allowing the PI stain to penetrate the disrupted cell and stain the genomic DNA, indicated by a red fluorescence intensity on the flow cytometer. Damaged cell membranes can be accompanied by the assumption that these cells no longer have metabolic activity and thus, are incapable of reproducing viably, hence the flow cytometry data was highly reliable to account for the loss in viability.

Example 5

Monitoring of chlorine treatment in gram-negative and gram-positive by PMA and DyeTox13 Green C-2 Azide-qPCR analysis

Next, E. coli, P. aeruginosa, E. faecalis, and B. sphaericus cells were exposed with chlorine to evaluate chlorine disinfection efficiency (General Procedure 2). As shown in Fig. 11 , all cells were exposed to three different concentrations of chlorine (10, 20, and 50 mg/mL). When gram-negative E. coli cells were treated with chlorine, the AC_t values from the PMA-qPCR assay showed an increase from 0.5 to 2.1 (Fig. 1 1A, white bars). Similarly, the AC_t values of DyeTox13 Green C-2 Azide-treated gram-negative cells also increased from 1.8 to 2.0 (Fig. 11 A, grey bars). Similar to the E. coli, when P. aeruginosa PA01 cells (see Fig. 1 1 B) were treated with 10 mg/mL chlorine, the AC_t from the PMA or DyeTox13 Green C-2 Azide-qPCR assays exhibited no differences. After being exposed to 20 mg/mL, however, the AC_t in both PMA and DyeTox13 Green C-2 Azide increased from 10 to 14. The effects of the PMA and DyeTox13 Green C-2 Azide-qPCR assays on E. faecalis cells are presented in Fig. 11C. A considerable difference in AC_t values were observed. However, the AC_t difference in DyeTox13 Green C-2 Azide-qPCR (7 to 8) was higher than PMA-qPCR (2 to 4). These results indicated that DyeTox13 Green C-2 Azide was more efficient at inhibiting PCR amplification than PMA. The AC_t values for viable B. sphaericus cells (Fig. 11 D) showed a considerable difference in AC_t values. Furthermore, the B. sphaericus results showed a similar tendency to the P. aeruginosa when comparing with AC_t values. As for the UV disinfectant results, the effect on DyeTox13 Green C-2 Azide treatment efficiency on gram-positive cells was greater than on the gram-negative cells.

Claims

Claims

1. A compound of formula I:

where:

R¹ is C alkyl and R² is H, or R¹ and R² together with the atoms that they are attached to form a 5- to 8-membered heterocyclic ring;

R³ represents H, Y(CH₂)_nN₃, or 0C(=0)CH₂N₃;

R⁴ represents Y(CH₂)_nN₃, (CH₂)_mN⁺(R⁷)₃ or aryl, where the aryl group is unsubstituted or substituted by halo, Ci-e alkyl or Y(CH₂)_nN₃;

R⁵ represents H, OR⁸ or Y(CH₂)_nN₃;

R⁶ represents H or OR⁸;

R⁷ and R⁸ independently represent at each occurrence Ci-e alkyl;

Y represents NR⁹ or O;

X represents one or more counter ions to balance the positive charge of the organic molecule; R⁹ represents H or Ci-e alkyl;

n is a number from 2 to 10;

m is a number from 2 to 10,

tautomers thereof, geometric isomers thereof and solvates thereof, provided that at least one of R³, R⁴, R⁵ contains an azide group.

2. The compound according to Claim 1 , wherein

R¹ is Ci-₃ alkyl and R² is H, or R¹ and R² together with the atoms that they are attached to form a 5- to 6-membered heterocyclic ring;

R³ represents H, Y(CH₂)_nN₃, or 0C(=0)CH₂N₃;

R⁴ represents Y(CH₂)_nN₃, (CH₂)_mN⁺(R⁷)₃ or aryl, where the aryl group is unsubstituted or substituted by halo, Ci-₃ alkyl or Y(CH₂)_nN₃; R⁵ represents H, OR⁸ or Y(CH₂)_nN₃;

R⁶ represents H or OR⁸;

R⁷ and R⁸ independently represent at each occurrence Ci-₃ alkyl;

Y represents NR⁹ or O;

R⁹ represents H or Ci-₃ alkyl;

n is a number from 2 to 5; and

m is a number from 2 to 5.

3. The compound according to Claim 1 or Claim 2, wherein

R¹ is CH3 and R² is H, or R¹ and R² together with the atoms that they are attached to form a 6-membered heterocyclic ring;

R³ represents H, Y(CH₂)nN₃, or 0C(=0)CH₂N₃;

R⁴ represents Y(CH₂)₂N₃, (CH₂)₂N⁺(R⁷)₃ or aryl, where the aryl group is unsubstituted or substituted by CH₃ or Y(CH₂)_nN₃;

R⁵ represents H, OCH₃ or Y(CH₂)_nN₃;

R⁶ represents H or OCH₃; and

Y represents NH or O.

4. The compound according to any one of the preceding claims, wherein only one of R³, R⁴ and R⁵ contains an azide group.

5. The compound according to any one of the preceding claims, wherein the compound of formula I is selected from the group consisting of:

(i)

PCT/SG2019/050143

6. The compound according to any one of the preceding claims, wherein the compound of formula I is:

7. A method of detecting and/or quantitating the presence of metabolically active microorganisms in a test sample, the method comprising:

(f) providing a test sample comprising microorganisms;

(g) contacting said microorganisms with a compound of any one of claims 1 to 6 for a period in the dark;

(h) exposing said microorganisms from (b) to light irradiation to activate said compound;

(i) amplifying a target region of DNA of said microorganisms from step (c) by a nucleic acid amplification method; and (j) comparing the level of amplified product from (d) with that of a control amount of said microorganism.

8. The method according to claim 7, wherein DNA is isolated from the exposed microorganisms from step (c) prior to amplification in step (d).

9. The method according to either one of the claims 7 or 8, wherein the microorganism is selected from one or more of the group consisting of bacteria, fungi and yeast.

10. The method according to any one of the claims 7 to 9, wherein the DNA in step (d) is amplified using a method selected from the group consisting of Polymerase chain reaction (PCR), Quantitative polymerase chain reaction (qPCR), Strand displacement amplification (SDA), Helicase-dependent amplification (HDA), Nicking enzyme amplification reaction (NEAR) and loop-mediated isothermal amplification (LAMP).

11. The method according to any one of claims 7 to 10, wherein the test sample is selected from the group comprising foodstuff, a biological sample, drinking water, industrial water, environmental water, wastewater, soil and clinical samples.

12. A microorganism metabolic activity test kit to detect and/or quantitate the presence of metabolically active microorganisms in a test sample, the kit comprising:

(c) a compound of any one of claims 1 to 6;

(d) amplification primers that target a region of a DNA of the said microorganisms, preferably, wherein at least one of the said primers is structurally and/or chemically modified from its corresponding natural nucleic acid.

13. The microorganism metabolic activity test kit of claim 12, wherein the microorganism is selected from one or more of the group consisting of bacteria, fungi and yeast.

14. The microorganism metabolic activity test kit of claim 12 or 13, wherein one or more structural and/or chemical modifications are selected from the group comprising the addition of tags, such as fluorescent tags, radioactive tags, biotin, a 5’ tail, the addition of phosphorothioate (PS) bonds, 2'-0-Methyl modifications and/or phosphoramidite C3 Spacers during synthesis.