Journal of Applied Microbiology 2001, 91, 1118±1130

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

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. Signi®cance 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 speci®c 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 de®nitions. In particular, the most recent revision of the de®nition of coliform bacteria in the UK is based on the possession of b-galactosidase (Anon. 1994). This de®nition has encouraged the development of new media and methodologies, based on presence±absence (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 7á8, which causes only partial dissociation, around 30%, to the highly ¯uorescent anion at the pH of the external growth medium, usually around pH 7á0 (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
ã 2001 The Society for Applied Microbiology

and the solution brought to re¯ux temperature. 1985). Sterile. Wild strains were isolated from pathological samples in the Microbiology Department. The progress of the reaction was followed by thin layer chromatography. all chemicals and solvents were obtained from Sigma-Aldrich Chemical Company Ltd. thus providing scope for synthesizing coumarins with a lower pKa (Goodwin and Kavanagh 1950. 7-hydroxy-4-methylcoumarin-b-D-galactoside and 7-hydroxycoumarin-4-acetic acid-b-D-galactoside. respectively. Central Public Health Laboratory Service. After cooling. To the stirred solution was added either dimethyl malonate (2á9 g) or diethyl malonate (3á5 g). involving condensation of a b-ketonic ester with a substituted resorcinol. as assays that include such substrates are expected to recover stressed organisms (Camper and McFeters 1979. the product was ®ltered and recrystallized from methanol. which was also the source for 7-hydroxy-4-methylcoumarin. It is well documented that derivatization of the coumarin molecule at various positions signi®cantly alters the deprotonation of the 7-hydroxyl group. 6-chloro-7hydroxy-4-methylcoumarin was prepared as follows. released by hydrolysis. In order to obtain bacterial suspensions with densities equivalent to particular McFarland Standard  rieux. Recrystallization from methanol gave the coumarin as a grey precipitate which was puri®ed by a second recrystallization to give white microcrystals (2á8 g). Basingstoke. Methyl 7-hydroxycoumarin-3-carboxylate and ethyl 7-hydroxycoumarin-3-carboxylate were prepared via a Knoevenagel condensation (Jones 1967) as follows. ¯at-bottomed microtitre trays (Bibby Sterilin Limited.D E T E C T I O N O F B A C T E R I A L b. The free acid was recrystallized from hot aqueous ethanol. UK. The solid residue was then separated. Fluorescence was measured using a Labtech Biolite F1 ¯uorescence microtitre plate reader (Labtech) with excitation and emission ®lters at 365 nm and 440 nm. Bacteriological media were obtained from LabM. Wolfbeis et al. Preparation of 3-acetyl-6-chloro-7-hydroxy-4methylcoumarin was from 4-chlororesorcinol and ethyl diacetoacetate. A 4á3 g sample of 4-chlororesorcinol was melted using a gentle heat into 3á3 g of ethyl acetoacetate to create a homogenous liquid. The absorbances of experimental media were measured using an Anthos 2001 spectrophotometric microtitre plate reader (Labtech International Limited. washed with water and air-dried. (1991). Freeman Hospital. The temperature was allowed to rise to ambient. followed by acidi®cation to pH 3á0. UK). MATERIALS AND METHODS Materials Unless otherwise stated. Uck®eld.G A L A C T O S I D A S E 1119 proportion of molecules in their anionic form at neutral pH. UK) were used throughout. Journal of Applied Microbiology. re¯ux was continued for 2±3 h. Morpholine (150 mg) and acetic acid (50 mg) were mixed together and added to the reaction mixture as catalyst. as required. The present study was undertaken to synthesize a range of ¯uorescent coumarin derivatives. any toxicity associated with such substrates will be of particular importance when used with environmental samples. 3-chloro-7-hydroxy4-methylcoumarin was prepared by condensation of resorcinol with ethyl 2-chloroacetoacetate. Although substrates based on 4-MU are used extensively in diagnostic microbiology. should not be inhibitory to microbial growth. Aberdeen. UK) at an absorption wavelength of 690 nm. Methods Synthesis of coumarins. This assay involved a modi®cation of the miniature multiple tube dilution method described by Hernandez et al. Preparation of 7-hydroxycoumarin-4-acetic acid was from resorcinol and diethyl acetonedicarboxylate. the toxicity of such substrates has not been examined critically. 7-hydroxycoumarin-3-carboxylic acid was obtained by mild alkaline hydrolysis of ethyl 7-hydroxycoumarin-3-carboxylate. For example. UK) or National Collections of Industrial and Marine Bacteria Limited (NCIMB. Calabrese and Bissonette 1990). and stirring was continued for 16 h before pouring the mixture into well-stirred ice-water. An important feature of any coumarin-based substrate is that the core molecule. London. Most of the coumarins were prepared using the essential Pechmann reaction (Sethna and Phadke 1953). Bacterial strains were obtained from the National Collection of Type Cultures (NCTC. Aberbargoed. The core molecule 7-hydroxy-4-methylcoumarin-3-propionic acid was prepared from resorcinol and diethyl 2-acetylglutarate. 1118±1130 . alongside 4-MU-GAL. Finally. The most suitable compounds were then glycosidated to form b-D-galactosides. Figure 1 and Table 1 illustrate the positions at which the core ã 2001 The Society for Applied Microbiology. and to evaluate their pKa and possible inhibitory effects on bacterial growth. 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. for the detection of coliforms in water samples. To the stirred solution was added 30 ml of ice-cold 75% sulphuric acid.4-dihydroxybenzaldehyde was dissolved in 15 ml anhydrous methanol. 91. The ester formed was hydrolysed by warming with dilute potassium hydroxide solution. Using the same basic method. Acidi®cation of the reaction mixture yielded the carboxylic acid that was crystallized from boiling water. Bury. UK) was values. A 2á8 g sample of 2. The most appropriate substrate was then applied in a rapid MPN-based assay. Furthermore. a Densimat (bioMe used in all experimental procedures.

5á4 mmol) was added in diffuse light and. All subsequent dilutions were carried out using sterile BHI broth. each stock coumarin solution was diluted (1 : 300) in a range of phosphate buffers over the pH range 4á0±8á0. Each suspension was then diluted in sterile BHI (1 : 1000) to a density of » 3 ´ 105 cfu ml±1. coli were used in this experiment. Values were averaged and the ¯uorescence was plotted against pH for each coumarin. strains were diluted to the same bacterial density. 0á032. resulting in the ®nal concentrations of coumarin and organism to be half of those listed above. 0á125. Synthesis of coumarinic galactosides. A more extensive investigation of growth inhibition was carried out with 4-MU. Active silver carbonate (1á5 g. 0á016. 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 ã 2001 The Society for Applied Microbiology. and the structure of the respective groups. as well as three wild strains of each of the three species (see Table 2). 5 mmol). Eight strains of E. Analysis of coumarins for ¯uorescence properties and toxicity. 1 Structure of 7-hydroxycoumarin (umbelliferone) molecule 7-hydroxycoumarin was derivatized. 0á125. 91. 4á0 mmol) was suspended in dry dichloromethane (15 ml) containing a crushed. Fig. 1á0. 0á008 and 0á004 mmol l±1. followed by a-acetobromo-D-galactose (2á06 g. Journal of Applied Microbiology. C H I L V E R S ET AL. to provide a ®nal maximum absorbance reading (24 h). seven wild strains and one type strain (NCTC 10418). respectively. 0á5. Appropriate coumarin-free and bacteria-free controls were included in each microtitre tray. including individual type strains of the three coliforms Escherichia coli (National Collection of Type Cultures 10418).6-collidine (1á5 ml) and stirring continued until the ester was dissolved. in intervals of 0á5 pH units.1120 K .4. The strains were cultivated on Columbia agar at 37°C for 18 h. F . resulting in ®nal concentrations of coumarin and numbers of organism of 0á5. in triplicate. which also included a DMSO solvent control prepared at the same concentrations as the coumarin stock solutions. Twelve coliform organisms were included in the toxicity assay. Plate counts were performed on Columbia agar to con®rm bacterial numbers. Stock 4-MU was diluted in BHI broth to achieve the following concentrations: 2á0. incorporating a broader range of concentrations. Absorbance at 690 nm was monitored at 30 min intervals for the initial 6 h of incubation at 37°C. The pH of each stock coumarin broth was checked prior to ®lter-sterilization and adjusted to pH 7á4 ‹ 0á2 if necessary. 1118±1130 . after 10 min. Five of the coumarin molecules were subsequently derivatized to form galactosides as follows. The reaction mixture was stirred for 2±3 days at Table 1 Groups able to derivatize 7-hydroxycoumarin at the positions labelled in Fig. Klebsiella pneumoniae (NCTC 10896) and Citrobacter freundii (NCTC 9750). activated 0á4 nm molecular sieve (200 mg). All strains were cultivated and harvested as above. Once dissolved. Aliquots of 50 ll were added to an equal volume of each coumarin dilution in triplicate. 0á1 mmol of each coumarin core molecule was weighed out and dissolved in 1 ml dimethylsulphoxide (DMSO). Methyl 7-hydroxycoumarin-3-carboxylate (0á88 g. To establish any potential inhibitory effects on the growth of coliform bacteria. Aliquots of 50 ll were added to an equal volume of coumarin broth. To establish the effect of pH on the ¯uorescence of each coumarin molecule. 0á25. each strain was then harvested and suspended in BHI broth to a density equivalent to a McFarland Standard of 1á0. with shaking. 0á25 and 0á0625 mmol l±1. namely 3 ´ 105 cfu ml±1. Trays were then incubated for a further 18 h at 37°C in the absence of shaking. each stock coumarin solution was diluted in brain heart infusion (BHI) broth to achieve the following concentrations: 1á0. 0á064. To the magnetically-stirred suspension was added 2. 0á03125 mmol l±1 and 1á5 ´ 105 cfu ml±1. Triplicate 100 ll aliquots of each buffered coumarin were added to microtitre trays and the ¯uorescence read.

1118±1130 . yielding 1á3 g of the acetylated glycoside. Addition of methanol caused crystallization of the protected galactoside. pneumoniae Citrobacter freundii C. 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. After ®ltration and phase separation. This was removed by vacuum ®ltration and dried to yield 1á5 g protected galactoside. pneumoniae Citrobacter freundii C. and washed with excess water to remove the core molecule. pneumoniae Kl. coli E. The same methodology was used to prepare ethyl 7-hydroxycoumarin3-carboxylate-b-D-galactoside (EHC-GAL). The synthetic procedure for 7-hydroxy-4-methylcoumarin-3-propionic acid-b-D-galactoside closely followed the previous example. potassium hydroxide (0á2 g. cloacae Enterobacter aerogenes Ent. freundii C. and in the following preparation. 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) 15±20°C in the dark. along with a further addition of a-acetobromo-D-galactose (1 g. It was deprotected using sodium methoxide/methanol as described above. freundii Enterobacter cloacae Ent. coli Klebsiella pneumoniae Kl. 5 mmol) was sus- pended in acetone (10 ml). washed with ether and dried in vacuo. the reaction mixture was poured into aqueous 0á4 mol l±1 hydrochloric acid (100 ml) and the product collected on a ®lter funnel. and to the stirred suspension was added potassium hydroxide (0á42 g. The residue was crystallized from hot methanol. excess Na+ ions were removed by agitation with ion exchange using Amberlite IR120H+ prior to isolation of the ®nal product. The tetraacetyl galactoside was deprotected as previously described. coli E. Analysis of toxicity of coumarinic galactosides and b-galactosidase activity with various coliform bacteria. as described previously. Journal of Applied Microbiology. After stirring for 16 h. After 48 h. An excess of alkali was required to maintain a pH between 10 and 12. Stirring was continued overnight and the suspension poured into 150 ml stirred ice-water. The organic layer was agitated with sodium carbonate (2á12 g. homogenous by thin layer chromatography.D E T E C T I O N O F B A C T E R I A L b. To this mixture was added aacetobromo-D-galactose (2á06 g. However. freundii C. 20 mmol) in water (10 ml) with the addition of Dowex Marathon ion exchanger. 5á0 mmol) in acetone (5 ml). The product was deacetylated in methanol-dichloromethane using sodium methoxide. 1994) was ã 2001 The Society for Applied Microbiology. The combined organic layers were dried using magnesium sulphate. The reaction mixture was ®ltered through a pad of coarse silica gel powder and eluted with aliquots of dichloromethane (100 ml total volume). and (ii) MPN assay Strains used for core coumarin/coumarin-galactoside analysis Escherichia coli E. 2á4 mmol) in acetone (5 ml). To prepare 6-chloro-7-hydroxy-4-methylcoumarinb-D-galactoside. Modi®ed Membrane Lauryl Sulphate Broth (mMLSB) was used for the ¯uorescence assay and for establishing whether the galactosides exhibited any inhibitory effect on bacterial growth. the organic layer was dried using magnesium sulphate and evaporated under reduced pressure. freundii Strains used for MPN assay Escherichia coli E. In this. The dichloromethane layer was washed with water. dried. Deprotection was performed by dissolving in a dichloromethane/methanol mixture with the addition of a catalytic quantity of methanolic sodium methoxide. Residual acetylated galactoside was extracted into dichloromethane and the solution dried and evaporated to yield a white mousse. Thin layer chromatography showed almost complete glycosidation to methyl 7-hydroxycoumarin-3-carboxylate-b-D-galactoside.G A L A C T O S I D A S E 1121 Table 2 Strains used for: (i) analysis of core coumarin and coumaringalactoside analysis. Diethyl ether was added and the precipitate was removed by vacuum ®ltration. the coumarin (1á05 g. the progress was followed by thin layer chromatography. 3á5 mmol) in water (2 ml) was added. The dried product amounted to 0á8 g. The extract was washed with three successive aliquots of 0á4 mol l±1 hydrochloric acid (100 ml) to remove collidine. After 36 h. MLSB (Anon. 7á5 mmol) in water (10 ml) to achieve dissolution. The sticky solid which separated was removed by decantation and then dissolved in 50 ml dichloromethane. coli Klebsiella pneumoniae Kl. Both 4-MU-GAL and 7-hydroxycoumarin-4-acetic acid were obtained commercially (SigmaAldrich Chemical Company Ltd). in which the core molecule was sparingly soluble. and was ®nally washed with water. pneumoniae Kl. ®ltered. the mixture of core molecule and protected galactoside was separated by extraction with dichlorormethane. and evaporated under reduced pressure. deprotection was complete with precipitation of the glycoside visibly evident. 91.

based on three doubling dilutions with 32 replicates per dilution. Only two core molecules showed a lower ¯uorescence at pH 7á0 than 4-MU. isopropylb-D-thiogalactoside (IPTG) was added at 60 mg l±1 to each substrate broth. 4-MU exhibited a minimal effect (data not shown) whereas growth was inhibited above this level. A total of 32 replicates of each dilution for each organism was included in this assay. The data clearly show some coumarin core molecules to be substantially less inhibitory to bacterial growth than 4-MU. Trays were incubated overnight (37°C). 0á34 g l±1). then read half-hourly for a further 5 h. was directly compared with 4-MU-GAL (1 mmol l±1. giving preliminary results at 24 h: trays were re-incubated for a further 24 h to give maximum counts. Ent. Figure 3 speci®cally illustrates the effect of pH on the ¯uoresence of each coumarin core molecule. the difference in ¯uorescence between ethyl 7-hydroxycoumarin-3-carboxylate and 4-MU was even more pronounced than at pH 7á0. Each substrate/IPTG broth was added to microtitre trays in 50 ll aliquots. For example. if necessary. MPN dilutions were prepared. At concentrations below 0á008 mmol l±1. Inoculated microtitre plates were incubated for a total of 11 h with shaking at 37°C. Trays were incubated at 37°C with shaking. As for the toxicity studies of the core molecules. plate counts were taken prior to the MPN assay to con®rm bacterial numbers. Fluorescence (365/ 440 nm) was read at time zero and again at 6 h. based on bacterial population estimates of 5 ´ 109 cfu ml±1. RESULTS Initial studies showed that. Six coumarin core molecules showed greater ¯uorescence at the same pH. the same 12 coliform organisms were harvested from Columbia agar after 18 h incubation at 37°C. Lauryl Tryptose Broth (LTB. F . at this pH. together with the ¯uorescence values from each of the core molecules at pH 7á0. Journal of Applied Microbiology. prepared in modi®ed format to contain no phenol red and no lactose. monitoring both absorbance (690 nm) and ¯uorescence (365/440 nm) hourly for 6 h. followed by ®lter-sterilization. Appropriate substrate-free and bacteriafree controls were included in each microtitre tray. to record an 18 h maximum reading of both ¯uorescence and absorbance. ã 2001 The Society for Applied Microbiology. followed by the addition of an equal volume of bacterial suspension. 1118±1130 . For example. the increase in absorbance at 300 min was only 46% of that produced by the growth control. with heating (to < 80°C) if necessary. indicating how poorly the ¯uorescence of 4-MU compares with other coumarin core molecules. Escherichia coli NCIMB 10213. 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. the pH of each substrate/IPTG broth was checked and altered to 7á4 ‹ 0á2. 100 and 50 cfu ml±1. both substrate broths incorporated IPTG (60 mg l±1) and were prepared as described earlier. An amount equivalent to 0á01 mmol of each substrate was weighed out and dissolved in 10 ml mMLSB. 1991) which uses a 96-well microtitre plate for the MPN assay. aerogenes NCIMB 10102 and Klebsiella pneumoniae NCTC 10896 were included in this assay. 91. For example. the ¯uorescence of 4-MU was 37% of the ¯uorescence exhibited by ethyl 7-hydroxycoumarin-3-carboxylate. Table 3 shows the effect of other coumarin core molecules at a concentration of 0á5 mmol l±1 on growth of a range of 12 coliforms (averaged results. without shaking. Citrobacter freundii NCTC 9750. with increasing concentration.1122 K . Anon. This was based on a miniaturized MPN procedure (Hernandez et al. Ethyl 7-hydroxycoumarin3-carboxylate-b-D-galactoside. reading ¯uorescence at 24 h to give maximum counts. Application of galactosides to MPN assay format. A range of doubling dilutions of each organism was prepared in sterile water to attain counts in the order of » 200. Once dissolved. along with one wild strain of each species. at 0á5 mmol l±1. coli at various concentrations of 4-MU. with substrate present at 0á5 mmol l±1. A modi®ed version of this procedure was used. EHC-GAL (1 mmol l±1. Microtitre trays containing standard LTB were incubated without shaking at 37°C. Plate counts were taken to con®rm bacterial numbers. Figure 2 shows a representative set of data for growth of E. C H I L V E R S ET AL. Aliquots of 100 ll of organism were added to 100 ll of each double-strength mLTB. up to a maximum of 1 mmol l±1. in BHI broth. to achieve MPN values for a total of 10 coliform organisms diluted to low inoculum densities (< 200 cfu ml)1). 1994) was modi®ed (mLTB) to contain no lactose or bromocresol purple and prepared at double strength. the ¯uorescence of 4-MU being 20% of that exhibited by ethyl 7-hydroxycoumarin-3-carboxylate. 0á4 g l±1). 30 mg l±1 IPTG and 3 ´ 106 cfu ml)1 bacterial density. resulting in ®nal concentrations of 0á5 mmol l±1 substrate. 4-MU was inhibitory to optimal growth of coliform bacteria. All experimental work was carried out in triplicate. Each strain was suspended in mMLSB to a density equivalent to a McFarland Standard of 1á0 and then diluted (1:50) in mMLSB to a suspension density of 6 ´ 106 cfu ml±1. 10 and 5 cfu per microtitre well. Enterobacter cloacae NCTC 11936. At pH 6á0. and the dilution series predicted counts in the order of » 20. Strains were cultivated on Columbia agar for 18 h at 37°C. Fluorescence data show that some coumarin core molecules were substantially more ¯uorescent than 4-MU at pH 7á0. Trays were then incubated overnight at 37°C. followed by inoculation into 10 ml BHI broth for a further 18 h at 37°C. 7-hydroxycoumarin3-carboxylic acid generated the same increase in absorbance as the control. based on absorbance change).

(d).G A L A C T O S I D A S E 1123 0·18 0·16 0·14 0·12 0·10 Abs (690 nm) Fig. (m). 1 mmol l±1. (h). (n). ã 2001 The Society for Applied Microbiology. control 0·08 0·06 0·04 0·02 0·00 0 30 60 90 120 150 180 210 240 270 300 −0·02 Time (min) Table 3 Fluorescence of 0á5 mmol l±1 of coumarin core molecules at pH 7á0 and the effect of each core molecule on coliform growth (averaged data from 12 coliform strains) Fluorescence at pH 7á0 (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. (j).D E T E C T I O N O F B A C T E R I A L b. 1118±1130 . 2 Growth of Escherichia coli (NCTC 10418) in the presence of various concentrations of 7-hydroxy-4-methylcoumarin (4-MU) in BHI broth. 0á008 mmol l±1. (s). 0á25 mmol l±1. 91. 0á5 mmol l±1. 0á125 mmol l±1. Journal of Applied Microbiology.

(d). 6-chloro-7-hydroxy4-methylcoumarin.1124 K . these data suggest that this substrate may have been slightly inhibitory to bacterial growth when compared with the novel substrates. (j). The results of the studies carried out with four newlysynthesized coumarinic galactosides produced no inhibitory effect on bacterial growth. EHC-GAL generated the maximum ¯uorescence of the coumarinic galactosides after 6 h incubation. whereas after 18 h of incubation. based on absorbance readings at 690 nm after overnight incubation (Table 4). 7-hydroxy-4methylcoumarin-3-propionic acid ã 2001 The Society for Applied Microbiology. Certain galactoside substrates generated substantially more ¯uorescence upon hydrolysis by the growing bacterial culture than other substrates. Although 4-MU-GAL performed reasonably well. four of the ®ve media containing novel substrates showed increases in absorbance identical to that of the growth control. (n). ethyl 7-hydroxycoumarin3-carboxylate. 7-hydroxycoumarin4-acetic acid. Journal of Applied Microbiology. resulted from the growth medium containing 4-MUGAL. These data con®rm 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. 91. (m). The lowest increase in absorbance. and all were better than 4-MU-GAL at 6 h. (s). with the average increase in absorbance in the presence of the substrate being 95% that of the growth control. and the ¯uorescence generated from the hydrolysis of 4-MU-GAL was only 27á7% (2836) of the 80 000 70 000 60 000 Fluorescence (365/440 nm) 50 000 40 000 30 000 20 000 10 000 0 4 4·5 5 5·5 6 6·5 7 7·5 8 pH Fig. observed at both incubation times. 3 Effect of pH of the ¯uorescence of various coumarin molecules at a concentration of 0á5 mmol l±1. Any coumarin core molecules that were less inhibitory after overnight incubation than 4-MU were selected for derivatization into b-D-galactoside substrates. (h). 7-hydroxy-4-methylcoumarin (4-MU). 1118±1130 . F . 3-chloro-7-hydroxy4-methylcoumarin. C H I L V E R S ET AL.

(h).G A L A C T O S I D A S E 1125 Table 4 Averaged data from 12 coliform organisms showing (i) the effect of each coumarinic galactoside at 0á5 mmol l±1 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. (s). 1118±1130 . Control. (n).ethyl 7-hydroxycoumarin-3-carboxylate-b-D-galactoside.D E T E C T I O N O F B A C T E R I A L b. (r). (m). 91. 4 Fluorescence generated from the hydrolysis of a range of coumarin galactosides (0á5 mmol l±1) by Citrobacter freundii (wild strain) in mMLSB. (j). (d). 7-hydroxycoumarin-4-acetic acid-b-D-galactoside. 6-chloro-7-hydroxy-4-methylcoumarin-b-D-galactoside. 7-hydroxycoumarin3-carboxylic acid-b-D-galactoside 5000 0 0 60 120 180 240 300 360 Time (min) −5000 ã 2001 The Society for Applied Microbiology. 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. 7-hydroxy-4-methylcoumarin-3-propionic acid-b-D-galactoside. 7-hydroxy-4-methylcoumarin-b-D-galactoside (4-MU-GAL). Journal of Applied Microbiology.

freundii (wild strain) over 6 h of incubation. This illustrates that with eight of the 10 coliform organisms used in this study. Journal of Applied Microbiology. with the exception of 7-hydroxy-4-methyl-3-propionic acid-b-D-galactoside. for two ã 2001 The Society for Applied Microbiology. coli NCIMB 10213 E. However.1126 K . 2000). 5 Comparison of the MPN values achieved with ®ve type strains and ®ve wild strains of various coliforms with two modi®cations of Lauryl Tryptose Broth (mLTB) after 660 min incubation at 37 °C. These results are typical of those obtained for all coliform strains. Although 6-chloro7-hydroxy-4-methylcoumarin-b-D-galactoside generated the maximum average ¯uorescence of the b-galactosidase substrates tested after 18 h incubation. mLTB containing 4-MU-GAL generated lower MPN values after 11 h incubation than in mlTB containing the newly-synthesized substrate EHC-GAL. NCTC 9750 (wild) NCTC (wild) aerogenes aerogenes pneumoniae pneumoniae NCIMB (wild) NCTC (wild) 11936 10102 10896 Fig. Kl. 7-hydroxy-4-methylcoumarinb-D-galactoside (4-MU-GAL) ¯uorescence value generated at 6 h from EHC-GAL (10252). cloacae Ent. Overall. The differences in ¯uorescence generation were less pronounced after 18 h of incubation. with an average ¯uorescence value greater than threefold that of 4-MU-GAL after 6 h incubation. 100 90 80 70 60 MPN ml −1 50 40 30 20 10 0 E. Kl. (j). Figure 4 shows representative ¯uorescence data generated by the hydrolysis of ®ve coumarinic galactosides by C. Ent. indicating its potential for detecting low numbers of target organisms more quickly than existing methodologies. freundii Ent. 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. Consequently. Ethyl 7-hydroxycoumarin-3-carboxylate-b-D-galactoside. F . 1118±1130 . in contrast to EHC-GAL (Table 4). 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. coli (wild) C. C H I L V E R S ET AL. freundii C. it was not selected for application in an MPN assay due to the relatively low ¯uorescence generated after 6 h incubation. for the 12 coliform organisms used in this investigation. Figure 5 shows a comparison of MPN values achieved with 10 coliform organisms in two ¯uorogenic modi®cations of mLTB after 11 h incubation. (h). cloacae Ent. although the average ¯uorescence generated upon hydrolysis of 4-MUGAL still compared poorly with that of most of the novel coumarin b-galactosidase substrates. 91. EHC-GAL yielded maximal ¯uorescence upon hydrolysis.

residual chlorine.D E T E C T I O N O F B A C T E R I A L b. 1999). 2000). the novel substrate generated higher MPN values than 4-MU-GAL with eight of the 10 organisms included in the assay. Coliforms. 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. 1118±1130 . to obtain a pH greater than 10 (George et al. 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 0á056 for the comparison between mLTB plus 4-MU-GAL and standard LTB only. an alkalinization step may be required in ¯uorogenic assays. Figure 7 shows the MPN values achieved after 24 h using both ¯uorogenic substrates in comparison with those ã 2001 The Society for Applied Microbiology. as shown in Fig. pneumoniae). and this may be of concern where 4-MU-GAL is used in rapid assay format. (s). can survive in drinking water for between 4 and 12 weeks. cloacae and Kl. a disadvantage of substrates based on 4-MU is that the maximum ¯uorescence of the reaction product requires an alkaline pH. Fluorescence is substantially quenched at pH levels below the pKa of the ¯uorophore. whereas even after 24 h of incubation. 91. Covert et al. Although the P-value for the mLTB plus 4-MU-GAL/standard LTB comparison is just above that required to determine a statistically signi®cant difference. pneumoniae). As with the 11 h data. As a result. since the pKa of 4-MU is around 8á0 (Goodwin and Kavanagh 1950). 6 A comparison of MPN values of Klebsiella pneumoniae (NCTC 10896) over 24 h incubation in the presence of 4-MU-GAL and EHCGAL. enabling optimal ¯uorescence at more acidic pH values. 1992). Moreover. Ethyl 7-hydroxycoumarin-3-carboxylate-b-D-galactoside (EHC-GAL). 1988. The ®nal MPN ml±1 value of 28 was reached by EHC-GAL at 630 min. DISCUSSION Fluorogenic substrates based on 4-MU have been widely used for b-galactosidase detection and coliform testing of water samples (Edberg et al. coli determinations (based on b-glucuronidase) in treated water systems (Clark 10 5 0 0 360 390 420 450 480 510 540 570 600 630 660 24h Fig. including E. the MPN value of 4-MUGAL was still rising and was 18% lower than that attained by EHC-GAL. the MPN value was highest in mLTB containing 4-MUGAL. Journal of Applied Microbiology. 3. depending on environmental conditions such as water temperature. 7-hydroxy-4-methylcoumarin-b-D-galactoside (4-MU-GAL) coliforms (wild strain of Ent. with some studies observing unacceptable levels of false-negative E.G A L A C T O S I D A S E 1127 30 25 20 15 achieved using the US standard medium LTB. pneumoniae (NCTC 10896) over the time period when samples were assayed half-hourly. coli. aerogenes and Kl. resulting in lower than optimal signals under most reaction conditions (Gee et al. In this study. usually involving the addition of concentrated alkali to the growth medium following enzymatic digestion of the substrate. Figure 6 shows the increase in MPN ml±1 for Kl. and lower values for the remaining two coliforms (wild strains of Ent. derivatization of the 4-MU core molecule at various positions resulted in shifted ¯uorescence±pH curves. the presence of other micro¯ora and exposure to solar ultraviolet radiation (Edberg et al. The ability of enzyme detection methods based on 4-MU to recover stressed organisms has been questioned. it was apparent that a positive MPN value (³ 1 cfu ml±1) was reached with EHC-GAL on average 1 h earlier than with 4-MU-GAL. However. When increases in MPN values over the incubation period were compared for each substrate. 2000). while the equivalent P-value for the equivalent comparison between EHC-GAL and standard LTB was 0á32. 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. (d).

C H I L V E R S ET AL. while the medium would then be further incubated to attain a quantitative result at 18 or 24 h. The use of enzyme-speci®c substrates has signi®cantly reduced the labour and time involved in processing water samples for coliform indicator bacteria. Kl. A ¯uorogenic detection system could indicate when a positive result ( ˆ `coliform present') was achieved so that the relevant action could be taken. 1992). freundii C. allowing remedial action to be taken (Sartory and Watkins 1999). which might indicate a treatment failure in a drinking water sample. might be responsible for such results (McFeters et al. 200 180 160 140 120 MPN ml −1 100 80 60 40 20 0 E. Factors such as these increase the potential applications of a substrate with a lower inhibitory effect than 4-MU. Ethyl 7-hydroxycoumarin-3carboxylate-b-D-galactoside (EHC-GAL). However. 91. 7-hydroxy-4-methylcoumarin-bD-galactoside (4-MU-GAL). Covert et al. F . This indicates the potential of this substrate for use in a rapid assay. NCIMB NCTC 9750 (wild) NCTC (wild) aerogenes aerogenes pneumoniae pneumoniae 10213 11936 NCIMB (wild) NCTC (wild) 10102 10896 Fig. resulting from disinfectants. (j). LTB et al. Journal of Applied Microbiology. Kl. LTB. 1991. to the growth of coliforms. A ¯uorogenic identi®cation system using a coumarinic galactoside such as this has the potential to identify both coliforms and E. The results of the present investigation suggest that the novel galactoside substrates described here are potentially more likely to recover organisms. 7 Comparison of the MPN values achieved with ®ve type strains and ®ve wild strains of various coliforms with unmodi®ed and two modi®cations of standard Lauryl Tryptose Broth after 24 h incubation at 37 °C. The 24 h incubation data presented here illustrate that the novel substrate EHC-GAL performed comparably with the US standard recommended medium. cloacae Ent. 1986). the signi®cance of this MPN assay is the time taken to detect a positive well containing the target coliform organism. It has been suggested that sublethal injury. more meaningful protection of public health would be achieved if results of coliform and E. Ent.1128 K . coli assays were available on the same day as the samples were collected. released when 4-MU-GAL is hydrolysed. 1118±1130 . coli E. (h). 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. ( ). Furthermore. Such differences may be further enhanced when the target organisms are sublethally injured. freundii Ent. coli (wild) C. cloacae Ent. for example. coli simultaneously by incorporating a b-glucuronidase substrate along with a b-galactosidase substrate. ã 2001 The Society for Applied Microbiology. as the core molecules released upon hydrolysis are measurably less inhibitory than 4-MU.

A. Journal of Applied Microbiology.A. 335±337.. D. In Chemical Methods in Prokaryotic Systematics ed. W. speci®c. (1991) Fluorogenic and chromogenic substrates used in bacterial diagnostics. G.J.M. Jones. Rankin. Archives of Biochemistry and Biophysics 27. Rice.S. W. Goodfellow. Rice. M. pp. The non-inhibitory effect of the novel core molecules will be an important factor when recovery of bacteria from natural waters is required. A. S. and Wolfrom. Roybal..P... G. Guibert. (1999) Fluorogenic substrates based on ¯uorinated umbelliferones for continuous assays of phosphatases and b-galactosidases. de®ned substrate technology for the simultaneous detection of total coliforms and Escherichia coli. P. (1991) Comparative study of commercial 4-methylumbelliferyl-b-D-glucuronide preparations with the Standard Methods membrane ®ltration faecal coliform test for the detection of Escherichia coli in water samples. McFeters 1990). A. Bhalgat.G. In Drinking Water Microbiology: Progress and Recent Developments ed. J. Conchie. S. Hernandez. Inc.L.M.. J. and Smith. particularly if rapid results are required. Goodwin. The hydrolysis of the substrate by b-galactosidase activity results in the release of core molecule into the growth medium.. pp. and McFeters. EHC-GAL. (1988) Rapid detection of total and faecal coliforms in water by enzymatic hydrolysis of 4-methylumbelliferone-b-D-galactoside. ethyl 7-hydroxycoumarin-3-carboxylate-b-D-galactoside.C. Reports on Public Health and Medical Subjects No. T.C.. Anon. 1528±1534.A. M. Kneifel. (1988) Rapid. J. M. L. Monatshefte fu È r Chemie 116. S. Wolfe. (2000) Use of enzymatic methods for rapid enumeration of coliforms in freshwaters.. 335±348. and Allen. occurrence and signi®cance of injured indicator bacteria in drinking water. Karlin.N. 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. and O'Donnell. G. Further work with the coumarinic b-D-galactoside substrates is needed to evaluate their performance with environmental water samples. C. In Methods in Carbohydrate Chemistry ed. M. Clarke. Milner.. R. In Organic Reactions Vol. Methods for the Examination of Water and Associated Materials. E. Allen. Inc.. Calabrese. 2118±2122. James.H.K. P. Berman. 98±104. One of these substrates. M. McFeters.P. Reviews in Medical Microbiology 4. Applied and Environmental Microbiology 56. E. 91. reduced bacterial toxicity.K..W. phosphatases and sulfatases. (1993) Chromogenic and ¯uorogenic indicators and substrates in diagnostic microbiology.L. Stewart.B. New York: Springer-Verlag. Scarpino.A. 41±48.H. E. McFeters. Gee. (1979) Chlorine injury and the enumeration of waterborne coliform bacteria. Johnson. 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. Sun.B. Water Research 25. Covert. Camper.W. which may have implications for the recovery of low numbers of bacteria from natural waters and environmental samples. J. (1963) Aryl glycopyranosides by the Koenigs-Knorr method.G A L A C T O S I D A S E 1129 The present study has shown that 4-MU may not be the most effective ¯uorescent label for b-galactosidase assay systems. Wiley and Sons. (1950) Fluorescence of coumarin derivatives as a function of pH. 1073±1078. 198±206. (1990) Enumeration. Mana®. 152±173. and Levvy.H.J. Berg. It has been shown here that a systematic approach to substrate development can be used to formulate coumarinic galactoside substrates with improved ¯uorescence and. Edberg. 565±580. O. M. J.F. S. and Kavanagh. New York and London: Academic Press Inc. Cope. and Olson. Microbiological Reviews 55. F.J.H.R.L.. Stelma. I. 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