3 carboxy coumarin....keep on trying!!

7/23/2019 3 Carboxy Coumarin....Keep on Trying!!

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Synthesis and evaluation of novel uorogenic substratesfor the detection of bacterial b-galactosidase

K.F. Chilvers1, J.D. Perry1, A.L. James2 and R.H. Reed21Department 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 . 2 0 0 1 .

Aims: A widely used coumarin derivative is 7-hydroxy-4-methylcoumarin-b-DD-galactoside

(4-methylumbelliferone-b-DD-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 ofcore 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 thesubstrate 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-hydroxy-

4-methylcoumarin, from practically non-uorescent esters

can indicate whether a particular microbial enzyme is

present in a sample. National guidelines for the microbio-

logical examination of water (Anon. 1994, 2000) include

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 glycosidederivatives 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 coum-

arin 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 greaterCorrespondence to: K.F. Chilvers, Department of Microbiology, Freeman

Hospital, Newcastle upon Tyne, UK.

2001 The Society for Applied Microbiology

Journal of Applied Microbiology 2001, 91, 11181130


2/13

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 depro-

tonation 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 sub-

strates 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 pKaand possible inhibitory effects on bacterial growth. The

most suitable compounds were then glycosidated to form

b-DD-galactosides. These novel uorogenic substrates were

then critically compared with 4-methylumbelliferone-b-DD-

galactoside (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-methylcouma-

rin, 7-hydroxy-4-methylcoumarin-b-DD-galactoside and

7-hydroxycoumarin-4-acetic acid-b-DD-galactoside. Bacterio-

logical media were obtained from LabM, Bury, UK.

Bacterial strains were obtained from the National Collec-

tion 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 patholo-

gical samples in the Microbiology Department, Freeman

Hospital. In order to obtain bacterial suspensions with

densities equivalent to particular McFarland Standard

values, a Densimat (bioMerieux, Basingstoke, UK) was

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 meas-

ured 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 wereprepared using the essential Pechmann reaction (Sethna and

Phadke 1953), involving condensation of a b-ketonic ester

with a substituted resorcinol. For example, 6-chloro-7-

hydroxy-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 toambient, 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 recrystal-

lization to give white microcrystals (28 g).

Using the same basic method, 3-chloro-7-hydroxy-

4-methylcoumarin was prepared by condensation of resor-

cinol with ethyl 2-chloroacetoacetate. The core molecule

7-hydroxy-4-methylcoumarin-3-propionic acid was pre-

pared from resorcinol and diethyl 2-acetylglutarate. The

ester formed was hydrolysed by warming with dilute

potassium hydroxide solution, followed by acidication topH 30. The free acid was recrystallized from hot aqueous

ethanol. Preparation of 3-acetyl-6-chloro-7-hydroxy-4-

methylcoumarin 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-hy-

droxycoumarin-3-carboxylate were prepared via a Knoeve-

nagel condensation (Jones 1967) as follows. A 28 g sample of

2,4-dihydroxybenzaldehyde was dissolved in 15 ml anhy-

drous 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-hydroxy-

coumarin-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 chroma-

tography. 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

D E T E C T I O N O F B A C T E R I A L b- G A L A C T O S I D A S E 1119

2001 The Society for Applied Microbiology, Journal of Applied Microbiology, 91, 11181130


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molecule 7-hydroxycoumarin was derivatized, and the struc-

ture of the respective groups.

Analysis of coumarins for uorescence propertiesand toxicity. To establish the effect of pH on theuorescence of each coumarin molecule, 01 mmol of each

coumarin core molecule was weighed out and dissolved in

1 ml dimethylsulphoxide (DMSO). Once dissolved, eachstock 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 uores-

cence 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 BHIbroth. 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 1 0. 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, 0

125, 0

03125 mmol l

1

and15 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 toachieve 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 formgalactosides as follows. Methyl 7-hydroxycoumarin-3-carb-

oxylate (088 g, 40 mmol) was suspended in dry dichlo-

romethane (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-DD-galactose (206 g,

5 mmol). The reaction mixture was stirred for 23 days at

R1 R2 R3

7-hydroxy-4-methylcoumarin (4-MU) CH3 H H

7-hydroxycoumarin-4-acetic acid CH2COOH H H

Ethyl 7-hydroxycoumarin-3-carboxylate H COOC2H5 H

3-chloro-7-hydroxy-4-methylcoumarin CH3 Cl H

6-chloro-7-hydroxy-4-methylcoumarin CH3 H Cl

Methyl 7-hydroxycoumarin-3-carboxylate H COOCH3 H

3-acetyl-6-chloro-7-hydroxy-4-methylcoumarin CH3 COCH3 Cl

7-hydroxycoumarin-3-carboxylic acid H COOH H

7-hydroxy-4-methylcoumarin-3-propionic acid CH3 C2H5COOH H

Table 1 Groups able to derivatize 7-hydro-xycoumarin at the positions labelled in Fig. 1

Fig. 1 Structure of 7-hydroxycoumarin (umbelliferone)

1120 K . F . C H I L V E R S ET AL.



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1520C in the dark. Thin layer chromatography showed

almost complete glycosidation to methyl 7-hydroxycouma-rin-3-carboxylate-b-DD-galactoside.

The reaction mixture was ltered through a pad of coarse

silica gel powder and eluted with aliquots of dichloro-

methane (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 ofmethanolic 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 methodo-

logy was used to prepare ethyl 7-hydroxycoumarin-

3-carboxylate-b-DD-galactoside (EHC-GAL).

To prepare 6-chloro-7-hydroxy-4-methylcoumarin-

b-DD-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 a-

acetobromo-DD-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-DD-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 ltra-tion 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-b-

DD-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 andevaporated 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-methylcouma-

rin-3-propionic acid-b-DD-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 aspreviously described. Both 4-MU-GAL and 7-hydroxy-

coumarin-4-acetic acid were obtained commercially (Sigma-

Aldrich Chemical Company Ltd).

Analysis of toxicity of coumarinic galactosidesandb-galactosidase activity with variouscoliform bacteria. Modied Membrane Lauryl SulphateBroth (mMLSB) was used for the uorescence assay and for

establishing whether the galactosides exhibited any inhibi-

tory effect on bacterial growth. MLSB (Anon. 1994) was

Table 2 Strains used for: (i) analysis of core coumarin and coumarin-

galactoside analysis; and (ii) MPN assay

Strains used for core coumarin/coumarin-galactoside analysis

Escherichia coli NCTC 10418

E. coli Wild type (FrhEco 1)E. coli Wild type (FrhEco 2)

E. coli Wild type (FrhEco 3)

Klebsiella pneumoniae NCTC 10896

Kl. pneumoniae Wild type (FrhKpn 1)



Citrobacter freundii NCTC 9750

C. freundii Wild type (FrhCfr 1)



Strains used for MPN assay

Escherichia coli NCIMB 10213E. coli Wild type (FrhEco 1)

Klebsiella pneumoniae NCTC 10896


Citrobacter freundii NCTC 9750


Enterobacter cloacae NCTC 11936

Ent. cloacae Wild type (FrhEcl 1)

Enterobacter aerogenes NCIMB 10102

Ent. aerogenes Wild type (FrhEae 1)




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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, isopropyl-

b-DD-thiogalactoside (IPTG) was added at 60 mg l1 to each

substrate broth; the pH of each substrate/IPTG broth waschecked 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

0

5 mmol l

1

substrate, 30 mg l

1

IPTG and 3

10

6

cfu ml

)1

bacterial density. Appropriate substrate-free and bacteria-

free 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. Thiswas 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

organismsdiluted to lowinoculum 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-hydroxycoumarin-

3-carboxylate-b-DD-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 investiga-tion 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 eachdilution 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. coliat 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 moleculesat 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-hydroxycoumarin-

3-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 coremolecules 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-hydroxycouma-

rin-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.




6/13

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 coliformstrains)

Fluorescence at pH 70

(relative units)

Relative growth*

(% control)

7-hydroxy-4-methylcoumarin (4-MU) 25994 557-hydroxycoumarin-4-acetic acid 31823 91

Ethyl 7-hydroxycoumarin-3-carboxylate 70043 75

3-chloro-7-hydroxy-4-methylcoumarin 20021 37

6-chloro-7-hydroxy-4-methylcoumarin 43287 68

Methyl 7-hydroxycoumarin-3-carboxylate 76883 50

3-acetyl-6-chloro-7-hydroxy-4-methylcoumarin 47880 49

7-hydroxycoumarin-3-carboxylic acid 56175 100

7-hydroxy-4-methylcoumarin-3-propionic acid 20564 94

Control (growth medium and solvent) 100

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

002

000

002

004

006

008

010

012

014

016

018

0 30 60 90 120 150 180 210 240 270 300

Time (min)

Abs(69

0nm)

Fig. 2 Growth of Escherichia coli (NCTC

10418) in the presence of various concentra-

tions 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




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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 incuba-

tion than 4-MU were selected for derivatization intob-DD-galactoside substrates.

The results of the studies carried out with four newly-

synthesized 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-MU-

GAL, whereas after 18 h of incubation, four of the ve

media containing novel substrates showed increases inabsorbance 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

0

10 000

20 000

30 000

40 000

50 000

60 000

70 000

80 000

4 45 5 55 6 65 7 75 8

pH

Fluorescence(365/440nm)

Fig. 3 Effect of pH of the uorescence of

various coumarin molecules at a concentration

of 05 mmol l1. (d), 7-hydroxy-4-methyl-

coumarin (4-MU); (s), 7-hydroxycoumarin-

4-acetic acid; (j), ethyl 7-hydroxycoumarin-

3-carboxylate; (h), 3-chloro-7-hydroxy-

4-methylcoumarin; (m), 6-chloro-7-hydroxy-

4-methylcoumarin; (n), 7-hydroxy-4-

methylcoumarin-3-propionic acid




8/13

Table 4 Averaged data from 12 coliform organisms showing (i) the effect of each coumarinic galactoside at 0 5 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)

Relative growth at

18 h* (% control)

Average uorescence

at 6 h (relative units)

Average uorescence

at 18 h (relative units)

7-hydroxy-4-methyl-3-propionic acid-b-DD-galactoside

97 100 300 13357

Ethyl 7-hydroxycoumarin-3-carboxylate-

b-G-galactoside (EHC-GAL)

95 100 10252 30861

6-chloro-7-hydroxy-4-methylcoumarin-

b-DD-galactoside

100 99 2267 32546

7-hydroxycoumarin-3-carboxylic acid-

b-DD-galactoside

100 100 6388 28113

7-hydroxy-4-methylcoumarin-

b-DD-galactoside (4-MU-GAL)

90 95 2836 25794

7-hydroxycoumarin-4-acetic acid-

b-DD-galactoside

92 100 858 29894

Control (growth medium) 100 100

*Based on absorbance increase at 690 nm.

5000

0

5000

10 000

15 000

20 000

25 000

30 000

0 60 120 180 240 300 360

Time (min)

Fluorescence(365/440nm)

Fig. 4 Fluorescence generated from the

hydrolysis of a range of coumarin galacto-sides (05 mmol l1) by Citrobacter freundii

(wild strain) in mMLSB. (d), Control; (s),

7-hydroxy-4-methylcoumarin-3-propionic

acid-b-DD-galactoside; (j), 7-hydroxycouma-

rin-4-acetic acid-b-DD-galactoside; (h),ethyl

7-hydroxycoumarin-3-carboxylate-b-DD-galac-

toside; (m), 6-chloro-7-hydroxy-4-methyl-

coumarin-b-DD-galactoside; (n),

7-hydroxy-4-methylcoumarin-b-DD-galactoside

(4-MU-GAL); (r), 7-hydroxycoumarin-

3-carboxylic acid-b-DD-galactoside




9/13

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-MU-

GAL 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-DD-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-chloro-

7-hydroxy-4-methylcoumarin-b-DD-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 selec-

ted for testing in an MPN assay format in a direct

comparison with 4-MU-GAL and the standard US recom-

mended 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 illus-

trates 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

0

10

20

30

40

50

60

70

80

90

100

E.coli

NCIMB

10213

E.coli(wild) C.freundiiNCTC 9750

C. freundii(wild)

Ent.cloacaeNCTC11936

Ent.cloacae(wild)

Ent.aerogenes

NCIMB10102

Ent.aerogenes

(wild)

Kl.pneumoniae

NCTC10896

Kl.pneumoniae

(wild)

MPN

ml

1

Fig. 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. (j

), Ethyl7-hydroxycoumarin-3-carboxylate-b-DD-galac-

toside; (h), 7-hydroxy-4-methylcoumarin-

b-DD-galactoside (4-MU-GAL)




10/13

coliforms (wild strain of Ent. cloacae and Kl. pneumoniae),

the MPN value was highest in mLTB containing 4-MU-

GAL. 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 withEHC-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-MU-

GAL 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

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 differencebetween 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 indi-

cation 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 beof 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 sub-

stantially quenched at pH levels below the pKa of the

uorophore, resulting in lower than optimal signals undermost 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 uores-

cencepH curves, enabling optimal uorescence at more

acidic pH values, as shown in Fig. 3. Such uorescent

characteristics offer the advantage of not requiring alkalini-

zation and potentially generating a larger signal for the assay

of b-galactosidase activity in a non-destructive continuousassay 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

0

5

10

15

20

25

30

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 EHC-

GAL. (d), Ethyl 7-hydroxycoumarin-3-carboxylate-b-DD-galactoside

(EHC-GAL); (s), 7-hydroxy-4-methylcoumarin-b-DD-galactoside

(4-MU-GAL)




11/13

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 thatthe 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 per-

formed 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.

0

20

40

60

80

100

120

140

160

180

200

E.coliNCIMB10213

E. coli(wild)C. freundiiNCTC 9750C. freundii(wild) Ent. cloacaeNCTC11936

Ent.cloacae(wild) Ent.aerogenesNCIMB10102

Ent.aerogenes(wild)

Kl.pneumoniaeNCTC10896

Kl.pneumoniae(wild)

MPN

ml

1

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-3-

carboxylate-b-DD

-galactoside (EHC-GAL);( ), 7-hydroxy-4-methylcoumarin-b-

DD-galactoside (4-MU-GAL); (h), LTB




12/13

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 tobe 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 devel-

opment 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-DD-ga-

lactoside, EHC-GAL, has shown superior activity to 4-MU-

GAL in preliminary studies with coliform bacteria. Further

work with the coumarinic b-DD-galactoside substrates is

needed to evaluate their performance with environmentalwater 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).

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3 carboxy coumarin....keep on trying!!

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