Journal of Applied Microbiology 2001, 91, 11181130

Synthesis and evaluation of novel uorogenic substrates for the detection of bacterial b-galactosidase
K.F. Chilvers1, J.D. Perry1, A.L. James2 and R.H. Reed2
1

Department of Microbiology, Freeman Hospital, and 2School of Applied and Molecular Sciences, University of Northumbria at Newcastle, Newcastle upon Tyne, UK

2001/9: received 7 March 2001, revised 25 June 2001 and accepted 25 June 2001

K . F . C H I L V E R S , J . D . P E R R Y , A . L . J A M E S A N D R . H . R E E D . 2001.

Aims: A widely used coumarin derivative is 7-hydroxy-4-methylcoumarin-b-D-galactoside (4-methylumbelliferone-b-D-galactoside; 4-MU-GAL). This galactoside is utilized as a substrate for the detection of the b-galactosidase activity of coliform bacteria in water analysis. The intense uorescence of coumarin-based molecules has enabled them to be incorporated into enzyme-based tests for the quantitative assay of indicator bacteria. The aim of this present study was to evaluate the potential of other coumarin derivatives, by synthesis of a selection of core coumarin molecules. Methods and Results: Several coumarin derivatives were found to be more promising than 4-MU, with ethyl-7-hydroxycoumarin-3-caboxylate (EHC) giving a combination of greater uorescence over a broad pH range and reduced growth inhibition with 12 representative coliform strains. On conversion to a b-galactoside derivative, EHC-GAL generated a more rapid uorescence than any other tested substrate. Conclusions: When tested in a broth assay format, based on most probable number (MPN), low numbers of coliforms were detected with EHC-GAL around 1 h earlier than with 4-MU-GAL. Signicance and Impact of the Study: The present study suggests that EHC-GAL should be evaluated as a substrate for the detection of coliforms in water analysis, due to a combination of the following favourable features: (i) reduced toxicity; (ii) increased uorescence; (iii) pH stability of uorescence; and (iv) rapid detection.

INTRODUCTION Substrates based on 4-methylumbelliferone (4-MU) have been used extensively for the detection of enzymes in diagnostic microbiology (Mana et al. 1991; Dealler 1993; James 1994). This is due to the ease of hydrolysis and intense uorescence generated on release of 4-MU from the substrate by specic enzymes (Berg and Fiksdal 1988; Shadix and Rice 1991; Brenner et al. 1993). The formation of the highly uorescent 4-MU, also known as 7-hydroxy4-methylcoumarin, from practically non-uorescent esters can indicate whether a particular microbial enzyme is present in a sample. National guidelines for the microbiological examination of water (Anon. 1994, 2000) include
Correspondence to: K.F. Chilvers, Department of Microbiology, Freeman Hospital, Newcastle upon Tyne, UK.

enzymatic characteristics of indicator organisms in standard denitions. In particular, the most recent revision of the denition of coliform bacteria in the UK is based on the possession of b-galactosidase (Anon. 1994). This denition has encouraged the development of new media and methodologies, based on presenceabsence (P-A) or most probable number (MPN), for example, utilizing glycoside derivatives of 4-MU in a broth-based assay (Edberg et al. 1988). An inherent disadvantage of 4-MU is its relatively high pKa value of 78, which causes only partial dissociation, around 30%, to the highly uorescent anion at the pH of the external growth medium, usually around pH 70 (Koller and Wolfbeis 1985; Wolfbeis et al. 1985). Hydroxylated coumarin molecules generate their maximum uorescence in their anionic form. Therefore, a major advantage would be to synthesize a coumarin with a lower pKa resulting in a greater
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proportion of molecules in their anionic form at neutral pH. It is well documented that derivatization of the coumarin molecule at various positions signicantly alters the deprotonation of the 7-hydroxyl group, thus providing scope for synthesizing coumarins with a lower pKa (Goodwin and Kavanagh 1950; Wolfbeis et al. 1985). An important feature of any coumarin-based substrate is that the core molecule, released by hydrolysis, should not be inhibitory to microbial growth. Although substrates based on 4-MU are used extensively in diagnostic microbiology, the toxicity of such substrates has not been examined critically. Furthermore, any toxicity associated with such substrates will be of particular importance when used with environmental samples, as assays that include such substrates are expected to recover stressed organisms (Camper and McFeters 1979; Calabrese and Bissonette 1990). The present study was undertaken to synthesize a range of uorescent coumarin derivatives, and to evaluate their pKa and possible inhibitory effects on bacterial growth. The most suitable compounds were then glycosidated to form b-D-galactosides. These novel uorogenic substrates were then critically compared with 4-methylumbelliferone-b-Dgalactoside (4-MU-GAL) with respect to uorescence generation upon hydrolysis and growth inhibition effects. The most appropriate substrate was then applied in a rapid MPN-based assay, alongside 4-MU-GAL, for the detection of coliforms in water samples. This assay involved a modication of the miniature multiple tube dilution method described by Hernandez et al. (1991). MATERIALS AND METHODS Materials Unless otherwise stated, all chemicals and solvents were obtained from Sigma-Aldrich Chemical Company Ltd, which was also the source for 7-hydroxy-4-methylcoumarin, 7-hydroxy-4-methylcoumarin-b-D-galactoside and 7-hydroxycoumarin-4-acetic acid-b-D-galactoside. Bacteriological media were obtained from LabM, Bury, UK. Bacterial strains were obtained from the National Collection of Type Cultures (NCTC, Central Public Health Laboratory Service, London, UK) or National Collections of Industrial and Marine Bacteria Limited (NCIMB, Aberdeen, UK). Wild strains were isolated from pathological samples in the Microbiology Department, Freeman Hospital. In order to obtain bacterial suspensions with densities equivalent to particular McFarland Standard rieux, Basingstoke, UK) was values, a Densimat (bioMe used in all experimental procedures. The absorbances of experimental media were measured using an Anthos 2001 spectrophotometric microtitre plate reader (Labtech International Limited, Uckeld, UK) at an

absorption wavelength of 690 nm. Fluorescence was measured using a Labtech Biolite F1 uorescence microtitre plate reader (Labtech) with excitation and emission lters at 365 nm and 440 nm, respectively. Sterile, at-bottomed microtitre trays (Bibby Sterilin Limited, Aberbargoed, UK) were used throughout. Methods Synthesis of coumarins. Most of the coumarins were prepared using the essential Pechmann reaction (Sethna and Phadke 1953), involving condensation of a b-ketonic ester with a substituted resorcinol. For example, 6-chloro-7hydroxy-4-methylcoumarin was prepared as follows. A 43 g sample of 4-chlororesorcinol was melted using a gentle heat into 33 g of ethyl acetoacetate to create a homogenous liquid. To the stirred solution was added 30 ml of ice-cold 75% sulphuric acid. The temperature was allowed to rise to ambient, and stirring was continued for 16 h before pouring the mixture into well-stirred ice-water. The solid residue was then separated, washed with water and air-dried. Recrystallization from methanol gave the coumarin as a grey precipitate which was puried by a second recrystallization to give white microcrystals (28 g). Using the same basic method, 3-chloro-7-hydroxy4-methylcoumarin was prepared by condensation of resorcinol with ethyl 2-chloroacetoacetate. The core molecule 7-hydroxy-4-methylcoumarin-3-propionic acid was prepared from resorcinol and diethyl 2-acetylglutarate. The ester formed was hydrolysed by warming with dilute potassium hydroxide solution, followed by acidication to pH 30. The free acid was recrystallized from hot aqueous ethanol. Preparation of 3-acetyl-6-chloro-7-hydroxy-4methylcoumarin was from 4-chlororesorcinol and ethyl diacetoacetate. Preparation of 7-hydroxycoumarin-4-acetic acid was from resorcinol and diethyl acetonedicarboxylate. Methyl 7-hydroxycoumarin-3-carboxylate and ethyl 7-hydroxycoumarin-3-carboxylate were prepared via a Knoevenagel condensation (Jones 1967) as follows. A 28 g sample of 2,4-dihydroxybenzaldehyde was dissolved in 15 ml anhydrous methanol. To the stirred solution was added either dimethyl malonate (29 g) or diethyl malonate (35 g), as required, and the solution brought to reux temperature. Morpholine (150 mg) and acetic acid (50 mg) were mixed together and added to the reaction mixture as catalyst; reux was continued for 23 h. After cooling, the product was ltered and recrystallized from methanol. Finally, 7-hydroxycoumarin-3-carboxylic acid was obtained by mild alkaline hydrolysis of ethyl 7-hydroxycoumarin-3-carboxylate. The progress of the reaction was followed by thin layer chromatography. Acidication of the reaction mixture yielded the carboxylic acid that was crystallized from boiling water. Figure 1 and Table 1 illustrate the positions at which the core

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Fig. 1 Structure of 7-hydroxycoumarin (umbelliferone)

molecule 7-hydroxycoumarin was derivatized, and the structure of the respective groups. Analysis of coumarins for uorescence properties and toxicity. To establish the effect of pH on the uorescence of each coumarin molecule, 01 mmol of each coumarin core molecule was weighed out and dissolved in 1 ml dimethylsulphoxide (DMSO). Once dissolved, each stock coumarin solution was diluted (1 : 300) in a range of phosphate buffers over the pH range 4080, in intervals of 05 pH units. Triplicate 100 ll aliquots of each buffered coumarin were added to microtitre trays and the uorescence read. Values were averaged and the uorescence was plotted against pH for each coumarin. To establish any potential inhibitory effects on the growth of coliform bacteria, each stock coumarin solution was diluted in brain heart infusion (BHI) broth to achieve the following concentrations: 10, 025 and 00625 mmol l1. The pH of each stock coumarin broth was checked prior to lter-sterilization and adjusted to pH 74 02 if necessary. All subsequent dilutions were carried out using sterile BHI broth. Twelve coliform organisms were included in the toxicity assay, including individual type strains of the three coliforms Escherichia coli (National Collection of Type Cultures 10418), Klebsiella pneumoniae (NCTC 10896) and Citrobacter freundii (NCTC 9750), as well as three wild strains of each of the three species (see Table 2). The strains were cultivated on Columbia agar at 37C for 18 h; each strain was then harvested and suspended in BHI broth to a density equivalent to a McFarland Standard of 10. Each

suspension was then diluted in sterile BHI (1 : 1000) to a density of 3 105 cfu ml1. Plate counts were performed on Columbia agar to conrm bacterial numbers. Aliquots of 50 ll were added to an equal volume of coumarin broth, in triplicate, resulting in nal concentrations of coumarin and numbers of organism of 05, 0125, 003125 mmol l1 and 15 105 cfu ml1, respectively. Appropriate coumarin-free and bacteria-free controls were included in each microtitre tray, which also included a DMSO solvent control prepared at the same concentrations as the coumarin stock solutions. Absorbance at 690 nm was monitored at 30 min intervals for the initial 6 h of incubation at 37C, with shaking. Trays were then incubated for a further 18 h at 37C in the absence of shaking, to provide a nal maximum absorbance reading (24 h). A more extensive investigation of growth inhibition was carried out with 4-MU, incorporating a broader range of concentrations. Stock 4-MU was diluted in BHI broth to achieve the following concentrations: 20, 10, 05, 025, 0125, 0064, 0032, 0016, 0008 and 0004 mmol l1. Eight strains of E. coli were used in this experiment, seven wild strains and one type strain (NCTC 10418). All strains were cultivated and harvested as above; strains were diluted to the same bacterial density, namely 3 105 cfu ml1. Aliquots of 50 ll were added to an equal volume of each coumarin dilution in triplicate, resulting in the nal concentrations of coumarin and organism to be half of those listed above. Synthesis of coumarinic galactosides. Five of the coumarin molecules were subsequently derivatized to form galactosides as follows. Methyl 7-hydroxycoumarin-3-carboxylate (088 g, 40 mmol) was suspended in dry dichloromethane (15 ml) containing a crushed, activated 04 nm molecular sieve (200 mg). To the magnetically-stirred suspension was added 2,4,6-collidine (15 ml) and stirring continued until the ester was dissolved. Active silver carbonate (15 g, 54 mmol) was added in diffuse light and, after 10 min, followed by a-acetobromo-D-galactose (206 g, 5 mmol). The reaction mixture was stirred for 23 days at
Table 1 Groups able to derivatize 7-hydroxycoumarin at the positions labelled in Fig. 1

R1 7-hydroxy-4-methylcoumarin (4-MU) 7-hydroxycoumarin-4-acetic acid Ethyl 7-hydroxycoumarin-3-carboxylate 3-chloro-7-hydroxy-4-methylcoumarin 6-chloro-7-hydroxy-4-methylcoumarin Methyl 7-hydroxycoumarin-3-carboxylate 3-acetyl-6-chloro-7-hydroxy-4-methylcoumarin 7-hydroxycoumarin-3-carboxylic acid 7-hydroxy-4-methylcoumarin-3-propionic acid CH3 CH2COOH H CH3 CH3 H CH3 H CH3

R2 H H COOC2H5 Cl H COOCH3 COCH3 COOH C2H5COOH

R3 H H H H Cl H Cl H H

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Table 2 Strains used for: (i) analysis of core coumarin and coumaringalactoside analysis; and (ii) MPN assay Strains used for core coumarin/coumarin-galactoside analysis Escherichia coli E. coli E. coli E. coli Klebsiella pneumoniae Kl. pneumoniae Kl. pneumoniae Kl. pneumoniae Citrobacter freundii C. freundii C. freundii C. freundii Strains used for MPN assay Escherichia coli E. coli Klebsiella pneumoniae Kl. pneumoniae Citrobacter freundii C. freundii Enterobacter cloacae Ent. cloacae Enterobacter aerogenes Ent. aerogenes NCIMB 10213 Wild type (FrhEco 1) NCTC 10896 Wild type (FrhKpn 1) NCTC 9750 Wild type (FrhCfr 1) NCTC 11936 Wild type (FrhEcl 1) NCIMB 10102 Wild type (FrhEae 1) NCTC 10418 Wild type (FrhEco 1) Wild type (FrhEco 2) Wild type (FrhEco 3) NCTC 10896 Wild type (FrhKpn 1) Wild type (FrhKpn 2) Wild type (FrhKpn 3) NCTC 9750 Wild type (FrhCfr 1) Wild type (FrhCfr 2) Wild type (FrhCfr 3)

1520C in the dark. Thin layer chromatography showed almost complete glycosidation to methyl 7-hydroxycoumarin-3-carboxylate-b-D-galactoside. The reaction mixture was ltered through a pad of coarse silica gel powder and eluted with aliquots of dichloromethane (100 ml total volume). The extract was washed with three successive aliquots of 04 mol l1 hydrochloric acid (100 ml) to remove collidine, and was nally washed with water. The combined organic layers were dried using magnesium sulphate, ltered, and evaporated under reduced pressure. The residue was crystallized from hot methanol, yielding 13 g of the acetylated glycoside. Deprotection was performed by dissolving in a dichloromethane/methanol mixture with the addition of a catalytic quantity of methanolic sodium methoxide; the progress was followed by thin layer chromatography. After 36 h, deprotection was complete with precipitation of the glycoside visibly evident. Diethyl ether was added and the precipitate was removed by vacuum ltration, washed with ether and dried in vacuo. The dried product amounted to 08 g. The same methodology was used to prepare ethyl 7-hydroxycoumarin3-carboxylate-b-D-galactoside (EHC-GAL). To prepare 6-chloro-7-hydroxy-4-methylcoumarinb-D-galactoside, the coumarin (105 g, 5 mmol) was sus-

pended in acetone (10 ml), and to the stirred suspension was added potassium hydroxide (042 g, 75 mmol) in water (10 ml) to achieve dissolution. To this mixture was added aacetobromo-D-galactose (206 g, 50 mmol) in acetone (5 ml). After stirring for 16 h, potassium hydroxide (02 g, 35 mmol) in water (2 ml) was added, along with a further addition of a-acetobromo-D-galactose (1 g, 24 mmol) in acetone (5 ml). Stirring was continued overnight and the suspension poured into 150 ml stirred ice-water. The sticky solid which separated was removed by decantation and then dissolved in 50 ml dichloromethane. The dichloromethane layer was washed with water. The organic layer was agitated with sodium carbonate (212 g, 20 mmol) in water (10 ml) with the addition of Dowex Marathon ion exchanger. After ltration and phase separation, the organic layer was dried using magnesium sulphate and evaporated under reduced pressure. Addition of methanol caused crystallization of the protected galactoside. This was removed by vacuum ltration and dried to yield 15 g protected galactoside. The product was deacetylated in methanol-dichloromethane using sodium methoxide, as described previously. Preparation of 7-hydroxycoumarin-3-caboxylic acid-bD-galactoside was by a standard Koenigs-Knorr (Conchie and Levvy 1963) reaction using acetone/aqueous potassium hydroxide as described above. An excess of alkali was required to maintain a pH between 10 and 12. After 48 h, the reaction mixture was poured into aqueous 04 mol l1 hydrochloric acid (100 ml) and the product collected on a lter funnel, dried, and washed with excess water to remove the core molecule. Residual acetylated galactoside was extracted into dichloromethane and the solution dried and evaporated to yield a white mousse, homogenous by thin layer chromatography. It was deprotected using sodium methoxide/methanol as described above. In this, and in the following preparation, excess Na+ ions were removed by agitation with ion exchange using Amberlite IR120H+ prior to isolation of the nal product. The synthetic procedure for 7-hydroxy-4-methylcoumarin-3-propionic acid-b-D-galactoside closely followed the previous example. However, the mixture of core molecule and protected galactoside was separated by extraction with dichlorormethane, in which the core molecule was sparingly soluble. The tetraacetyl galactoside was deprotected as previously described. Both 4-MU-GAL and 7-hydroxycoumarin-4-acetic acid were obtained commercially (SigmaAldrich Chemical Company Ltd). Analysis of toxicity of coumarinic galactosides and b-galactosidase activity with various coliform bacteria. Modied Membrane Lauryl Sulphate Broth (mMLSB) was used for the uorescence assay and for establishing whether the galactosides exhibited any inhibitory effect on bacterial growth. MLSB (Anon. 1994) was

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prepared in modied format to contain no phenol red and no lactose. An amount equivalent to 001 mmol of each substrate was weighed out and dissolved in 10 ml mMLSB, with heating (to < 80C) if necessary. Once dissolved, isopropylb-D-thiogalactoside (IPTG) was added at 60 mg l1 to each substrate broth; the pH of each substrate/IPTG broth was checked and altered to 74 02, if necessary, followed by lter-sterilization. As for the toxicity studies of the core molecules, the same 12 coliform organisms were harvested from Columbia agar after 18 h incubation at 37C. Each strain was suspended in mMLSB to a density equivalent to a McFarland Standard of 10 and then diluted (1:50) in mMLSB to a suspension density of 6 106 cfu ml1. Plate counts were taken to conrm bacterial numbers. Each substrate/IPTG broth was added to microtitre trays in 50 ll aliquots, followed by the addition of an equal volume of bacterial suspension, resulting in nal concentrations of 05 mmol l1 substrate, 30 mg l1 IPTG and 3 106 cfu ml)1 bacterial density. Appropriate substrate-free and bacteriafree controls were included in each microtitre tray. All experimental work was carried out in triplicate. Trays were incubated at 37C with shaking, monitoring both absorbance (690 nm) and uorescence (365/440 nm) hourly for 6 h. Trays were then incubated overnight at 37C, without shaking, to record an 18 h maximum reading of both uorescence and absorbance. Application of galactosides to MPN assay format. This was based on a miniaturized MPN procedure (Hernandez et al. 1991) which uses a 96-well microtitre plate for the MPN assay. A modied version of this procedure was used, based on three doubling dilutions with 32 replicates per dilution, to achieve MPN values for a total of 10 coliform organisms diluted to low inoculum densities (< 200 cfu ml)1). Lauryl Tryptose Broth (LTB, Anon. 1994) was modied (mLTB) to contain no lactose or bromocresol purple and prepared at double strength. Ethyl 7-hydroxycoumarin3-carboxylate-b-D-galactoside, EHC-GAL (1 mmol l1, 04 g l1), was directly compared with 4-MU-GAL (1 mmol l1, 034 g l1); both substrate broths incorporated IPTG (60 mg l1) and were prepared as described earlier. Standard LTB (prepared at double strength to include lactose and bromocresol purple) was used in the investigation to compare MPN values achieved in the uorogenic assay with those from a medium based on US standard methods. Escherichia coli NCIMB 10213, Citrobacter freundii NCTC 9750, Enterobacter cloacae NCTC 11936, Ent. aerogenes NCIMB 10102 and Klebsiella pneumoniae NCTC 10896 were included in this assay, along with one wild strain of each species. Strains were cultivated on Columbia agar for 18 h at 37C, followed by inoculation into 10 ml BHI broth for a further 18 h at 37C. MPN dilutions were prepared, based on bacterial population estimates of 5 109 cfu ml1,

in BHI broth; plate counts were taken prior to the MPN assay to conrm bacterial numbers. A range of doubling dilutions of each organism was prepared in sterile water to attain counts in the order of 200, 100 and 50 cfu ml1. Aliquots of 100 ll of organism were added to 100 ll of each double-strength mLTB. A total of 32 replicates of each dilution for each organism was included in this assay, and the dilution series predicted counts in the order of 20, 10 and 5 cfu per microtitre well, with substrate present at 05 mmol l1. Inoculated microtitre plates were incubated for a total of 11 h with shaking at 37C. Fluorescence (365/ 440 nm) was read at time zero and again at 6 h, then read half-hourly for a further 5 h. Trays were incubated overnight (37C), reading uorescence at 24 h to give maximum counts. Microtitre trays containing standard LTB were incubated without shaking at 37C, giving preliminary results at 24 h: trays were re-incubated for a further 24 h to give maximum counts. RESULTS Initial studies showed that, with increasing concentration, 4-MU was inhibitory to optimal growth of coliform bacteria. Figure 2 shows a representative set of data for growth of E. coli at various concentrations of 4-MU, up to a maximum of 1 mmol l1. At concentrations below 0008 mmol l1, 4-MU exhibited a minimal effect (data not shown) whereas growth was inhibited above this level. For example, at 05 mmol l1, the increase in absorbance at 300 min was only 46% of that produced by the growth control. Table 3 shows the effect of other coumarin core molecules at a concentration of 05 mmol l1 on growth of a range of 12 coliforms (averaged results, based on absorbance change), together with the uorescence values from each of the core molecules at pH 70. The data clearly show some coumarin core molecules to be substantially less inhibitory to bacterial growth than 4-MU. For example, 7-hydroxycoumarin3-carboxylic acid generated the same increase in absorbance as the control. Fluorescence data show that some coumarin core molecules were substantially more uorescent than 4-MU at pH 70. For example, at this pH, the uorescence of 4-MU was 37% of the uorescence exhibited by ethyl 7-hydroxycoumarin-3-carboxylate. Six coumarin core molecules showed greater uorescence at the same pH, indicating how poorly the uorescence of 4-MU compares with other coumarin core molecules. Only two core molecules showed a lower uorescence at pH 70 than 4-MU. Figure 3 specically illustrates the effect of pH on the uoresence of each coumarin core molecule. At pH 60, the difference in uorescence between ethyl 7-hydroxycoumarin-3-carboxylate and 4-MU was even more pronounced than at pH 70, the uorescence of 4-MU being 20% of that exhibited by ethyl 7-hydroxycoumarin-3-carboxylate.

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018

016

014

012

010

Abs (690 nm)

Fig. 2 Growth of Escherichia coli (NCTC 10418) in the presence of various concentrations of 7-hydroxy-4-methylcoumarin (4-MU) in BHI broth. (d), 1 mmol l1; (s), 05 mmol l1; (j), 025 mmol l1; (h), 0125 mmol l1; (m), 0008 mmol l1; (n), control

008

006

004

002

000 0 30 60 90 120 150 180 210 240 270 300

002

Time (min)

Table 3 Fluorescence of 05 mmol l1 of coumarin core molecules at pH 70 and the effect of each core molecule on coliform growth (averaged data from 12 coliform strains)

Fluorescence at pH 70 (relative units) 7-hydroxy-4-methylcoumarin (4-MU) 7-hydroxycoumarin-4-acetic acid Ethyl 7-hydroxycoumarin-3-carboxylate 3-chloro-7-hydroxy-4-methylcoumarin 6-chloro-7-hydroxy-4-methylcoumarin Methyl 7-hydroxycoumarin-3-carboxylate 3-acetyl-6-chloro-7-hydroxy-4-methylcoumarin 7-hydroxycoumarin-3-carboxylic acid 7-hydroxy-4-methylcoumarin-3-propionic acid Control (growth medium and solvent) 25994 31823 70043 20021 43287 76883 47880 56175 20564

Relative growth* (% control) 55 91 75 37 68 50 49 100 94 100

*Based on absorbance increase at 690 nm after 6 h incubation.

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These data conrm that substituted coumarin molecules had been synthesized which were both substantially more uorescent than 4-MU and substantially less inhibitory to the growth of coliform organisms. Any coumarin core molecules that were less inhibitory after overnight incubation than 4-MU were selected for derivatization into b-D-galactoside substrates. The results of the studies carried out with four newlysynthesized coumarinic galactosides produced no inhibitory effect on bacterial growth, based on absorbance readings at 690 nm after overnight incubation (Table 4). Although 4-MU-GAL performed reasonably well, with the average increase in absorbance in the presence of the substrate being 95% that of the growth control, these data suggest that this

substrate may have been slightly inhibitory to bacterial growth when compared with the novel substrates. The lowest increase in absorbance, observed at both incubation times, resulted from the growth medium containing 4-MUGAL, whereas after 18 h of incubation, four of the ve media containing novel substrates showed increases in absorbance identical to that of the growth control, and all were better than 4-MU-GAL at 6 h. Certain galactoside substrates generated substantially more uorescence upon hydrolysis by the growing bacterial culture than other substrates. EHC-GAL generated the maximum uorescence of the coumarinic galactosides after 6 h incubation, and the uorescence generated from the hydrolysis of 4-MU-GAL was only 277% (2836) of the

80 000

70 000

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Fluorescence (365/440 nm)

50 000

40 000

30 000

20 000

10 000

0 4 45 5 55 6 65 7 75 8

pH

Fig. 3 Effect of pH of the uorescence of various coumarin molecules at a concentration of 05 mmol l1. (d), 7-hydroxy-4-methylcoumarin (4-MU); (s), 7-hydroxycoumarin4-acetic acid; (j), ethyl 7-hydroxycoumarin3-carboxylate; (h), 3-chloro-7-hydroxy4-methylcoumarin; (m), 6-chloro-7-hydroxy4-methylcoumarin; (n), 7-hydroxy-4methylcoumarin-3-propionic acid

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Table 4 Averaged data from 12 coliform organisms showing (i) the effect of each coumarinic galactoside at 05 mmol l1 on coliform growth and (ii) the uorescence generated as a result of hydrolysis of the substrates by bacterial b-galactosidase activity at 6 h and 18 h Relative growth at 6 h* (% control) 7-hydroxy-4-methyl-3-propionic acidb-D-galactoside Ethyl 7-hydroxycoumarin-3-carboxylateb-G-galactoside (EHC-GAL) 6-chloro-7-hydroxy-4-methylcoumarinb-D-galactoside 7-hydroxycoumarin-3-carboxylic acidb-D-galactoside 7-hydroxy-4-methylcoumarinb-D-galactoside (4-MU-GAL) 7-hydroxycoumarin-4-acetic acidb-D-galactoside Control (growth medium) *Based on absorbance increase at 690 nm. 97 95 100 100 90 92 100 Relative growth at 18 h* (% control) 100 100 99 100 95 100 100 Average uorescence at 6 h (relative units) 300 10252 2267 6388 2836 858 Average uorescence at 18 h (relative units) 13357 30861 32546 28113 25794 29894

30 000

25 000

20 000

Fluorescence (365/440 nm)

15 000

10 000

Fig. 4 Fluorescence generated from the hydrolysis of a range of coumarin galactosides (05 mmol l1) by Citrobacter freundii (wild strain) in mMLSB. (d), Control; (s), 7-hydroxy-4-methylcoumarin-3-propionic acid-b-D-galactoside; (j), 7-hydroxycoumarin-4-acetic acid-b-D-galactoside; (h),ethyl 7-hydroxycoumarin-3-carboxylate-b-D-galactoside; (m), 6-chloro-7-hydroxy-4-methylcoumarin-b-D-galactoside; (n), 7-hydroxy-4-methylcoumarin-b-D-galactoside (4-MU-GAL); (r), 7-hydroxycoumarin3-carboxylic acid-b-D-galactoside

5000

0 0 60 120 180 240 300 360

Time (min)
5000

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100

90

80

70

60

MPN ml

50

40

30

20

10

0
E. coli NCIMB 10213 E. coli (wild) C. freundii C. freundii Ent. cloacae Ent. cloacae Ent. Ent. Kl. Kl. NCTC 9750 (wild) NCTC (wild) aerogenes aerogenes pneumoniae pneumoniae NCIMB (wild) NCTC (wild) 11936 10102 10896

Fig. 5 Comparison of the MPN values achieved with ve type strains and ve wild strains of various coliforms with two modications of Lauryl Tryptose Broth (mLTB) after 660 min incubation at 37 C. (j), Ethyl 7-hydroxycoumarin-3-carboxylate-b-D-galactoside; (h), 7-hydroxy-4-methylcoumarinb-D-galactoside (4-MU-GAL)

uorescence value generated at 6 h from EHC-GAL (10252). The differences in uorescence generation were less pronounced after 18 h of incubation, although the average uorescence generated upon hydrolysis of 4-MUGAL still compared poorly with that of most of the novel coumarin b-galactosidase substrates, with the exception of 7-hydroxy-4-methyl-3-propionic acid-b-D-galactoside. Figure 4 shows representative uorescence data generated by the hydrolysis of ve coumarinic galactosides by C. freundii (wild strain) over 6 h of incubation. These results are typical of those obtained for all coliform strains. Overall, for the 12 coliform organisms used in this investigation, EHC-GAL yielded maximal uorescence upon hydrolysis, with an average uorescence value greater than threefold that of 4-MU-GAL after 6 h incubation. Although 6-chloro7-hydroxy-4-methylcoumarin-b-D-galactoside generated the maximum average uorescence of the b-galactosidase substrates tested after 18 h incubation, it was not selected

for application in an MPN assay due to the relatively low uorescence generated after 6 h incubation, in contrast to EHC-GAL (Table 4). Consequently, EHC-GAL was selected for testing in an MPN assay format in a direct comparison with 4-MU-GAL and the standard US recommended medium (Anon. 2000). The objective of the MPN-based assay was to compare the rate at which coliform bacteria could be detected by monitoring hydrolysis of EHC-GAL in comparison with 4-MU-GAL. Figure 5 shows a comparison of MPN values achieved with 10 coliform organisms in two uorogenic modications of mLTB after 11 h incubation. This illustrates that with eight of the 10 coliform organisms used in this study, mLTB containing 4-MU-GAL generated lower MPN values after 11 h incubation than in mlTB containing the newly-synthesized substrate EHC-GAL, indicating its potential for detecting low numbers of target organisms more quickly than existing methodologies. However, for two

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30

25

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achieved using the US standard medium LTB. As with the 11 h data, the novel substrate generated higher MPN values than 4-MU-GAL with eight of the 10 organisms included in the assay, and lower values for the remaining two coliforms (wild strains of Ent. aerogenes and Kl. pneumoniae). Moreover, comparisons of the difference between each uorogenic substrate in mLTB medium and the standard LTB medium using a one-sided paired t-test gave a P-value of 0056 for the comparison between mLTB plus 4-MU-GAL and standard LTB only, while the equivalent P-value for the equivalent comparison between EHC-GAL and standard LTB was 032. Although the P-value for the mLTB plus 4-MU-GAL/standard LTB comparison is just above that required to determine a statistically signicant difference, the results give an indication that MPN values obtained using 4-MU-GAL as a uorogenic substrate may be somewhat lower than those obtained with US standard LTB medium, and this may be of concern where 4-MU-GAL is used in rapid assay format. DISCUSSION Fluorogenic substrates based on 4-MU have been widely used for b-galactosidase detection and coliform testing of water samples (Edberg et al. 1988; Covert et al. 1992). However, a disadvantage of substrates based on 4-MU is that the maximum uorescence of the reaction product requires an alkaline pH, since the pKa of 4-MU is around 80 (Goodwin and Kavanagh 1950). Fluorescence is substantially quenched at pH levels below the pKa of the uorophore, resulting in lower than optimal signals under most reaction conditions (Gee et al. 1999). As a result, an alkalinization step may be required in uorogenic assays, usually involving the addition of concentrated alkali to the growth medium following enzymatic digestion of the substrate, to obtain a pH greater than 10 (George et al. 2000). In this study, derivatization of the 4-MU core molecule at various positions resulted in shifted uorescencepH curves, enabling optimal uorescence at more acidic pH values, as shown in Fig. 3. Such uorescent characteristics offer the advantage of not requiring alkalinization and potentially generating a larger signal for the assay of b-galactosidase activity in a non-destructive continuous assay format. Coliforms, including E. coli, can survive in drinking water for between 4 and 12 weeks, depending on environmental conditions such as water temperature, residual chlorine, the presence of other microora and exposure to solar ultraviolet radiation (Edberg et al. 2000). The ability of enzyme detection methods based on 4-MU to recover stressed organisms has been questioned, with some studies observing unacceptable levels of false-negative E. coli determinations (based on b-glucuronidase) in treated water systems (Clark

10

0 0 360 390 420 450 480 510 540 570 600 630 660 24h

Fig. 6 A comparison of MPN values of Klebsiella pneumoniae (NCTC 10896) over 24 h incubation in the presence of 4-MU-GAL and EHCGAL. (d), Ethyl 7-hydroxycoumarin-3-carboxylate-b-D-galactoside (EHC-GAL); (s), 7-hydroxy-4-methylcoumarin-b-D-galactoside (4-MU-GAL)

coliforms (wild strain of Ent. cloacae and Kl. pneumoniae), the MPN value was highest in mLTB containing 4-MUGAL. When increases in MPN values over the incubation period were compared for each substrate, it was apparent that a positive MPN value ( 1 cfu ml1) was reached with EHC-GAL on average 1 h earlier than with 4-MU-GAL. Figure 6 shows the increase in MPN ml1 for Kl. pneumoniae (NCTC 10896) over the time period when samples were assayed half-hourly. The nal MPN ml1 value of 28 was reached by EHC-GAL at 630 min, whereas even after 24 h of incubation, the MPN value of 4-MUGAL was still rising and was 18% lower than that attained by EHC-GAL. Figure 7 shows the MPN values achieved after 24 h using both uorogenic substrates in comparison with those

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1 100 80 60 40

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E. coli E. coli (wild) C. freundii C. freundii Ent. cloacae Ent. cloacae Ent. Ent. Kl. Kl. NCIMB NCTC 9750 (wild) NCTC (wild) aerogenes aerogenes pneumoniae pneumoniae 10213 11936 NCIMB (wild) NCTC (wild) 10102 10896

Fig. 7 Comparison of the MPN values achieved with ve type strains and ve wild strains of various coliforms with unmodied and two modications of standard Lauryl Tryptose Broth after 24 h incubation at 37 C. (j), Ethyl 7-hydroxycoumarin-3carboxylate-b-D-galactoside (EHC-GAL); ( ), 7-hydroxy-4-methylcoumarin-bD-galactoside (4-MU-GAL); (h), LTB

et al. 1991; Covert et al. 1992). It has been suggested that sublethal injury, resulting from disinfectants, might be responsible for such results (McFeters et al. 1986). Factors such as these increase the potential applications of a substrate with a lower inhibitory effect than 4-MU. The results of the present investigation suggest that the novel galactoside substrates described here are potentially more likely to recover organisms, as the core molecules released upon hydrolysis are measurably less inhibitory than 4-MU, released when 4-MU-GAL is hydrolysed, to the growth of coliforms. Such differences may be further enhanced when the target organisms are sublethally injured. The use of enzyme-specic substrates has signicantly reduced the labour and time involved in processing water samples for coliform indicator bacteria. However, more meaningful protection of public health would be achieved if results of coliform and E. coli assays were available on the same day as the samples were collected, allowing remedial action to be taken (Sartory and Watkins 1999). Higher MPN

values were achieved within 11 h of incubation when using mLTB plus EHC-GAL for eight of the 10 coliform organisms included in this trial. This indicates the potential of this substrate for use in a rapid assay. Furthermore, the signicance of this MPN assay is the time taken to detect a positive well containing the target coliform organism, which might indicate a treatment failure in a drinking water sample. A uorogenic detection system could indicate when a positive result ( `coliform present') was achieved so that the relevant action could be taken, while the medium would then be further incubated to attain a quantitative result at 18 or 24 h, for example. The 24 h incubation data presented here illustrate that the novel substrate EHC-GAL performed comparably with the US standard recommended medium, LTB. A uorogenic identication system using a coumarinic galactoside such as this has the potential to identify both coliforms and E. coli simultaneously by incorporating a b-glucuronidase substrate along with a b-galactosidase substrate.

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The present study has shown that 4-MU may not be the most effective uorescent label for b-galactosidase assay systems, particularly if rapid results are required. This is principally because the uorescence exhibited by 4-MU is low in comparison with other coumarin molecules at the pH of most growth media. In addition, 4-MU has been shown to be somewhat inhibitory to bacterial growth, which may have implications for the recovery of low numbers of bacteria from natural waters and environmental samples. It has been shown here that a systematic approach to substrate development can be used to formulate coumarinic galactoside substrates with improved uorescence and, based on core molecule studies, reduced bacterial toxicity. One of these substrates, ethyl 7-hydroxycoumarin-3-carboxylate-b-D-galactoside, EHC-GAL, has shown superior activity to 4-MUGAL in preliminary studies with coliform bacteria. Further work with the coumarinic b-D-galactoside substrates is needed to evaluate their performance with environmental water samples. A potential advantage of these novel substrates is that studies showed the corresponding core molecules to be less inhibitory to bacterial growth than 4-MU. The hydrolysis of the substrate by b-galactosidase activity results in the release of core molecule into the growth medium. The non-inhibitory effect of the novel core molecules will be an important factor when recovery of bacteria from natural waters is required, as such bacteria may be sublethally stressed and less able to cope with inhibitory components of the growth medium (McFeters et al. 1986; McFeters 1990). REFERENCES
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