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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
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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 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
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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)
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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)
Kl. pneumoniae Wild type (FrhKpn 2)
Kl. pneumoniae Wild type (FrhKpn 3)
Citrobacter freundii NCTC 9750
C. freundii Wild type (FrhCfr 1)
C. freundii Wild type (FrhCfr 2)
C. freundii Wild type (FrhCfr 3)
Strains used for MPN assay
Escherichia coli NCIMB 10213E. coli Wild type (FrhEco 1)
Klebsiella pneumoniae NCTC 10896
Kl. pneumoniae Wild type (FrhKpn 1)
Citrobacter freundii NCTC 9750
C. freundii Wild type (FrhCfr 1)
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.
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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
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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
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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)
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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)
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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
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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 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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