Journal of Microbiological Methods 79 (2009) 139–155

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Journal of Microbiological Methods

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j m i c m e t h

Review

Enzymatic substrates in microbiology☆

Sylvain Orenga a,⁎, Arthur L. James b, Mohammed Manafi c, John D. Perry b,d, David H. Pincus e
a
Research & Development Microbiology, bioMérieux, 3 route de Port Michaud, 38 390 La Balme-les-Grottes, France
b
Department of Applied Sciences, University of Northumbria, Newcastle upon Tyne, United Kingdom
c
Hygiene-Institute, Medical University of Vienna, Austria
d
Department of Microbiology, Freeman Hospital, Newcastle upon Tyne, United Kingdom
e
Research & Development Microbiology, bioMérieux, Saint Louis, MO, United States

a r t i c l e i n f o a b s t r a c t

Article history: Enzymatic substrates are powerful tools in biochemistry. They are widely used in microbiology to study
Received 19 May 2009 metabolic pathways, to monitor metabolism and to detect, enumerate and identify microorganisms.
Received in revised form 24 July 2009 Synthetic enzymatic substrates have been customized for various microbial assays, to detect an expanding
Accepted 3 August 2009
range of both new enzymatic activities and target microorganisms.
Available online 11 August 2009
Recent developments in synthetic enzymatic substrates with new spectral, chemical and biochemical
Keywords:
properties allow improved detection, enumeration and identification of food-borne microorganisms, clinical
Chromogenic medium pathogens and multi-resistant bacteria in various sample types. In the past 20 years, the range of synthetic
Detection enzymatic substrates used in microbiology has been markedly extended supporting the development of new
Identification multi-test systems (e.g., Microscan, Vitek 2, Phoenix) and chromogenic culture media. The use of such
Synthetic enzymatic substrate substrates enables an improvement in time to detection and specificity over conventional tests that employ
natural substrates.
In the era of intense developments in molecular biology, phenotypic tests involving enzymatic substrates
remain useful to analyse both simple and complex samples. Such tests are applicable to diagnostic and
research laboratories all over the world.
© 2009 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
M
. etabolic substrates, from natural to synthetic substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
2. Synthetic enzymatic substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
2.1. Fluorogenic substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
2.2. Chromogenic substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
2.3. Luminogenic substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
2.4. Secondary reaction based substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
2.5. Suicide substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
3. Microbial enzymatic activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
3.1. Hydrolases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
3.2. Nitroreductases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
3.3. Luciferases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
4. Enzymatic substrates in identification (ID) systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
5. Application of enzyme substrates in culture media for microbiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
5.1. Chromogenic media for clinical microbiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
5.1.1. Urinary tract pathogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
5.1.2. Salmonella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
5.1.3. S. aureus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
5.1.4. Differentiation of yeasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148

☆ Sponsor: Pr. A. Van Belkum — Erasmus Universiteit Medical Centre, Department of Medical Microbiology and Infectious Diseases, Room L-248, 's-Gravendijkwal 230, 3015 CE
Rotterdam, The Netherlands, Email: mailto:a.vanbelkum@erasmusmc.nl.
⁎ Corresponding author. Tel.: +33 4 74 95 25 43; fax: +33 4 74 95 26 43.
E-mail address: sylvain.orenga@eu.biomerieux.com (S. Orenga).

0167-7012/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.mimet.2009.08.001
140 S. Orenga et al. / Journal of Microbiological Methods 79 (2009) 139–155

5.1.5. Group B Streptococcus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148

5.1.6. Antibiotic-resistant bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
5.1.7. P. aeruginosa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
5.2. Fluorogenic and chromogenic media in food and water microbiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
5.2.1. E. coli and coliforms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
5.2.2. E. coli O157:H7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
5.2.3. Salmonella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
5.2.4. Shigella spp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
5.2.5. C. sakazakii . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
5.2.6. Y. enterocolitica . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
5.2.7. Vibrio spp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
5.2.8. Enterococci . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
5.2.9. C. perfringens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
5.2.10. L. monocytogenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
5.2.11. B. cereus and Bacillus thuringiensis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
5.2.12. Bacillus anthracis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
Disclosure statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150

1. Introduction lower toxicity at pH 7.0 (Chilvers et al., 2001). Consequently, after 6 h

Metabolic substrates, from natural to synthetic substrates
To simplify the study of metabolic activities, it is often advantageous
to use synthetic metabolic substrates that can provide an easily measured
signal such as a variation of absorbance or fluorescence, or to detect an
individual enzymatic activity in a complex metabolic pathway. Such
metabolic substrates are used in a wide range of fields and recent reviews
have focused on their use in the screening and optimisation of industrial
enzymes (Reymond et al., 2009) or in histochemistry (Kiernan, 2007). In
biochemistry and enzymology studies, the follow-up of enzymatic
activity can be based on secondary reactions or rely on separation and
sophisticated analytical instruments (Zhong et al., 1999). However, when
a high number of samples or enzymatic reactions have to be tested in a
short period of time, this is often not practical and it is easier to rely on
direct detection of the enzymatic products.
Synthetic substrates have been used to study microbial enzymatic
activities since the early 20th century (Aizawa, 1939). The first ones
were based on nitrophenol or nitroaniline (Aizawa, 1939, Lederberg,
1950). However, the background colour of most microbial culture
media is close to the yellow colour of these compounds (Manafi and
Kneifel, 1990). Moreover, the colour of nitrophenol is dramatically
reduced at acid pH. Also, colorimetric detection of naphthol or
naphthylamine based substrates relies on an end-point reaction with
a diazonium salt (Monget, 1975), which is not adapted to kinetic
analyses. Different groups of enzymatic substrates are more suited to
certain applications depending on a) the targeted enzymatic activity,
b) the mode of detection, and c) the type of reactional medium
(Table 1).
2. Synthetic enzymatic substrates
2.1. Fluorogenic substrates
incubation, the relative fluorescence produced by coliform bacteria grow-
ing in the presence of 4-MU-β-galactoside was only 28% of that generated
in the presence of EHC-β-galactoside. Similarly, when enterococci were
incubated with EHC-β-glucoside, the relative fluorescence was tenfold
higher than that produced with 4-MU-β-glucoside after 7 h incubation
(Perry et al., 2006a).
As 4-MU tends to diffuse away from bacterial cells, other substrates
may be preferred to demonstrate the localisation of enzymatic activity
based on fluorescence detection. For example, Huang et al. (1998) used
ELF97-phosphate to study the alkaline phosphatase expression of cells
within Klebsiella pneumoniae colonies and biofilm in response to phos-
phate starvation. In activated sludge samples, a similar phosphatase
substrate, 2-(5′-chloro-2′-phosphoryloxyphenyl)-4-[3]-quinazolinone,
allowed the distinction of three types of colonies: non-fluorescent ones
(i.e., no phosphatase activity), fluorescence limited to the colonies
(i.e., intracellular phosphatase activity) and fluorescent colonies sur-
rounded by a fluorescent halo (i.e., extracellular phosphatase activity)
(Van Ommen Kloeke et al., 1999). To alleviate problems with diffusion of
fluorescein, Zhang et al. (1991) synthesized lipophilic derivatives of
fluorescein-di-β-D-galactopyranoside, so the released fluorophore was
retained in the cells, allowing quantitation of β-galactosidase in yeast
cells.
Fluorogenic substrates have been designed with ‘intramolecular
quenching’ whereby a fluorophore is bound in close proximity of a
chromophore. Before hydrolysis, the chromophore quenches the
fluorescence but subsequent cleavage of any bond situated between
the two interacting groups, results in a marked increase in fluorescence
(Carmel et al., 1977). The enzymatically cleaved peptide bond of
intramolecularly quenched fluorogenic substrates could be similar to
the one of the natural substrate. Consequently, such substrates are very
useful to determine the true characteristics of peptidase. However, this
is not true when the assay mixture contains contaminating fluoro-
phores or quenchers (Vencill et al., 1985).

Fluorogenic substrates are widely used in microbiology. Most

of them are based on the fluorescent coumarin heterocycle, either as
4-methylumbelliferone (4-MU), e.g., for detection of glycosidases and
phosphatases, or as 7-amino-4-methylcoumarin for the detection of pep-
tidases (Goodfellow et al., 1990). However, the fluorescence of the 4-MU
is markedly reduced at low pH. 3-cyano-4-trifluoromethylumbelliferone
having a lower pKa than 4-MU, is better adapted for the design of
substrates for enzymatic assay at low pH (Markaryan and Voznyi, 1992).
Similarly, ethyl 7-hydroxycoumarin-3-carboxylate (EHC) has a relative
fluorescence five times higher than that of 4-MU at pH 6.0 and a slightly
2.2. Chromogenic substrates
Indoxyl based substrates initially developed for histochemistry
(Barrnett and Seligman, 1951) are widely used in microbiology
because the indigoid dye formed upon oxidation of liberated indoxyl
contrasts strongly from the colour of microbial media and precipitates
within colonies. Depending on the substituents on the indole ring, the
colour of the indigoid dyes ranges from blue to red (Sadler and
Warren, 1956, Kiernan, 2007).
 S. Orenga et al. / Journal of Microbiological Methods 79 (2009) 139–155
As the indigoid dyes formed upon intracellular hydrolysis of
indoxyl based substrates remain inside cells, they can be used for
single cell characterization (Poulsen et al., 1997).
Bainbridge et al. (1991) tried to develop substrates with higher
water solubility and lower cost than their indoxyl equivalent. However,
their proposed 2-(2-(4-(β-D-galactopyranosyloxy)-3-methoxyphenyl)-
vinyl)-3-methylbenzothiazolium toluene-4-sulphonate had to be used
with either filter paper or nitrocellulose membranes and in a limited pH
range. The application on colonies of a filter paper impregnated with the
enzymatic substrate was also required for the lipase substrates based on
5-(4-hydroxy-3,5-dimethoxyphenylmethylene)-2-thioxothiazoline-3-
acetic acid (Miles et al., 1992). Cooke et al. (2002) overcame this
limitation in a chromogenic medium for Candida with a closely related
substrate targeting hexosaminidase activity: 4-(2-[4-(2-acetamido-2-
deoxy-β-D-glucopyranosyloxy)-3-methoxyphenyl]-vinyl)-1-(propan-
3-yl-oate)-quinolium bromide (VLPA-GlcNAc). On their medium, more
yeast species hydrolysed the hexosaminidase substrate to produce pink
to red colonies than on chromogenic media incorporating 5-bromo-4-
chloro-3-indoxyl-N-acetyl-β-D-glucosaminide.
To overcome some limitations of indoxyl based substrates and
especially the costs and the requirement of aerobic incubation, James
et al. (2000a) synthesized p-naphtholbenzein-β-D-galactopyranoside
(pNB-gal). When incorporated in agar media, β-galactosidase pro-
ducing colonies were pink. For similar reasons, James et al. (2000b)
synthesized alizarin-β-D-galactoside (Aliz-gal). Upon hydrolysis, the
generated alizarin chelates with ferrous or aluminium ions forming
brightly coloured complexes.
When tested on strains of Enterobacteriaceae in the presence
of isopropyl-β-D-thiogalactopyranoside (IPTG), the sensitivity of
pNB-gal was equivalent to that of X-β-D-galactopyranoside (X-gal)
except for some strains of Serratia and Yersinia enterocolitica (James
et al., 2000a). With Aliz-gal, the sensitivity was slightly higher than
with X-gal for some strains of Serratia and Yersinia (James et al.,
2000b).
To detect β-glucuronidase activity of colonies of Escherichia coli,
James and Yeoman (1988) used 8-hydroxyquinoline-β-D-glucuronide.
The released 8-hydroxyquinoline chelates with ferrous ions present
in the medium generating an intense black pigmentation, but the
sensitivity was lower than that observed with 4-MU-β-D-glucuronide.
Black pigmentation is also obtained with the use of substrates based on
3,4-cyclohexenoesculetin (James et al., 1996, 1997), 3′,4′-dihydroxy-
flavone (Butterworth et al., 2004) and 3-hydroxyflavone (Perry et al.,
2006a). In those cases, the sensitivity was similar to that obtained with
indoxyl substrates for similar enzymatic activities.
Aminophenyl-acridine based substrates can be used for peptidase
detection (James et al., 2007). The 9-(4′-aminophenyl)-acridines are pale
yellow, but when protonated either as an acridinium salt or
upon addition of acetic acid after incubation, they turn red. Anderson
et al. (2008) used such novel chromogenic peptidase substrates based on
9-(4′-aminophenyl)-10-methylacridinium salts for direct detection of
microbial peptidase in colonies on agar plate media. Some of them appear
useful for differentiation among enterobacteria species. Chromogenic
peptidase substrates can also be based on derivatives of 7-amino-
phenoxazin-3-one (resorufamine, Zaytsev et al., 2008). The 1-pentyl
substituted derivative (pentylresorufamine, PRF) localises in the bacterial
cells, producing purple colonies on agar plate media (Fig. 1), but the L-
alanyl-PRF was inhibitory to Gram-positive bacteria.
2.3. Luminogenic substrates
Luminescence enables detection with high sensitivity and wide
dynamic range. The free enzyme assay of β-D-galactosidase based on
the chemiluminogenic substrate o-aminophthalylhydrazide-β-D-galacto-
side has a sensitivity 10 times higher than the fluorometric method based
on 4-MU-β-D-galactoside and 100 times higher than the colorimetric
method based on o-nitrophenyl-β-D-galactoside (Nakazono et al., 1992).
141
Ichibangase et al. (2004) designed a chemiluminescence assay for lipase
using the lauric acid ester of 2-(4-hydroxyphenyl)-4,5-diphenylimidazole
(HDI) as a substrate. The generated HDI acts as an enhancer of the
chemiluminescence reaction of luminol–hydrogen peroxide–horseradish
peroxidase. While this approach was successful for the assay of lipase
from Candida cylindracea and from porcine pancreas, it seems limited to
assay free enzyme rather than analyse whole living cells. To increase the
sensitivity of the detection of β-galactosidase activity from coliform cells,
Van Poucke and Nelis (1995 introduced a permeabilization step with
polymyxin B between the induction of cellular enzymatic activity and the
measurement of luminescence produced in the presence of a chloro
derivative of 3-{4-methoxyspiro [1,2-dioxetane-3,2′-tricyclo(3.3.1.13,7)
decan]-yl}phenyl-β-D-galactopyranoside. The phosphatidylinositol-
phospholipase C activity from culture supernatant of Bacillus cereus
was detected with a luminogenic substrate based on an adamantane–
dioxetane–phenyl group (Ryan et al., 1993).
The bioluminogenic substrate D-luciferin-o-β-galactopyranoside is
cleaved by β-galactosidase to release luciferin. This luciferin serves as
a substrate for luciferase (Geiger et al., 1992). For microorganism
analysis, it is combined with a detergent that lyses cells to release the
β-galactosidase present (de Almeida et al., 2008).
2.4. Secondary reaction based substrates
In some cases, the detection of enzymatic activity still relies on
detection of the natural metabolic product. This is the case for
tryptophanase that metabolises tryptophan to generate indole. The
indole may be detected by reaction with various aldehydes such as 4-
dimethylaminobenzaldehyde (DMABA), 4-dimethylaminocinnamalde-
hyde (DMACA), or 2-methoxy-4-dimethylaminobenzaldehyde (James
et al., 1986).
Due to low sensitivity of L-alanyl-p-nitroanilide for detecting the
D-alanyl-D-alanyl dipeptidase VanX from vancomycin-resistant entero-
cocci, Anissimova et al. (2003) developed an assay based on D-alanyl-α-
(R)-phenylthioglycine. The released α-phenylthioglycine was detected
upon reaction with 5,5′-dithio-bis-(2-nitrobenzoic acid) (Ellman's re-
agent) and could be continuously monitored spectrophotometrically.
2.5. Suicide substrates
As in oncology, microbiologists use some enzymatic activities to
inhibit specifically bacteria expressing those activities. While the
toxicity of the substrate is very low, one of the products of the
metabolism is highly toxic. It is useful especially with intracellular
enzymatic activities such as the β-galactosidase of E. coli (Park et al.,
1976). The application of this technology has been further extended
by the application of phosphonopeptides (Perry et al., 2002).
3. Microbial enzymatic activities
3.1. Hydrolases
Most of the synthetic enzymatic substrates used in microbiology are
substrates targeting hydrolases (Fig. 2). In particular, a wide range of
glycosidases, has been exploited as enzymatic targets (Manafi et al.,
1991). Substrates for β-ribofuranosidase appear useful for differentia-
tion between Y. enterocolitica, which does not hydrolyse these sub-
strates, and most other enterobacteria, which are positive (Butterworth
et al., 2004). A complex glycoside, 5-bromo-4-chloro-3-indoxyl-5′-
acetamido-3′,5′-dideoxy-α-D-glycero-D-galacto-2-nonulopyranosido-
nic acid, is hydrolysed by a neuraminidase from Clostridium perfringens
producing an insoluble indigoid dye upon aerobic oxidation (Fujii et al.,
1993). However, as C. perfringens requires an anaerobic atmosphere, it is
not adapted to direct detection of growing C. perfringens strains.
Deoxyribonuclease activity is expressed by Staphylococcus aureus
but not by Staphylococcus epidermidis. This could be used to differentiate
 142
Table 1
Examples of chromogenic and fluorogenic synthetic enzymatic substrates adapted to liquid or solid support applications and according to different groups of enzymatic activities.

S. Orenga et al. / Journal of Microbiological Methods 79 (2009) 139–155

 S. Orenga et al. / Journal of Microbiological Methods 79 (2009) 139–155
a Pócsi et al., 1990; b Armstrong et al., 2001; c Pearson et al., 1963; d Rothe et al., 1992.

143
144 S. Orenga et al. / Journal of Microbiological Methods 79 (2009) 139–155
Fig. 1. Hydrolysis of β-alanyl pentylresorufamine by colonies of Pseudomonas
aeruginosa after 72 h incubation.
both species with adapted substrates such as 5-bromo-4-chloro-3-
indoxyl-thymidine-3′-phosphate (Wolf et al., 1969).
Peptidases are useful in differentiation of bacteria (Goodfellow
et al., 1987, Manafi et al., 1991). L-Alanine aminopeptidase activity can
differentiate between Gram-negative and Gram-positive bacteria and
this has been demonstrated using L-alanine-7-amino-4-methylcou-
marin (Manafi and Kneifel, 1990) and, more recently, 9-(4′-N-[L-Ala-L-
Ala-L-Ala]aminophenyl)-10-methylacridinium (Anderson et al., 2008).
In contrast, β-alanine aminopeptidase activity is expressed by a more
limited range of Gram-negative bacteria, and β-alanyl-PRF appeared to
be a useful substrate to differentiate Pseudomonas aeruginosa colonies
on a culture medium (Zaytsev et al., 2008).
While most of the substrates for detection of peptidases are based
on L-amino acids, D-alanine-p-nitroanilide may be used to differen-
tiate Listeria monocytogenes (negative) from the other Listeria species
(positive) (Clark and McLaughlin, 1997).
Detection of carboxypeptidase can be achieved by the use of N-
benzoyl-amino acids. The generated amino acids then react with
ninhydrin. With N-benzoyl-L-histidine, it is possible to differentiate
Acinetobacter baumannii and Acinetobacter calcoaceticus (both posi-
tive) from most other Gram-negative bacteria (Perry et al., 1998).
When enzymes hydrolyse peptide bonds between two defined
amino acids, the intramolecularly quenched fluorogenic substrates are
well adapted. This approach has been successfully used by Fleminger
et al. (1982) to detect aminopeptidase P from E. coli. Combined with flow
cytometry, it allowed sorting of E. coli cells with active OmpT, a surface-
displayed protease, from cells with no OmpT (Olsen et al., 2000).
To overcome price and availability limitations of nitrocefin and
pyridine-2-azo-p-dimethylaniline cephalosporin (PADAC), a chromo-
genic substrate derived from cephalothin named CENTA (Bebrone
et al., 2001) can be used for detection of every type of β-lactamase
except for the CphA metallo-β-lactamase. However, due to the
absorption spectrum of the leaving group, it is not adapted for direct
detection of β-lactamase producing colonies on agar plates. Cefesone
Fig. 2. Hydrolysis of synthetic enzymatic substrates.
(S1, Sutton et al., 1995) is an alternative substrate for β-lactamases.
Positive reactions are detected by a change of colour from light yellow
to red. Results are very similar to those obtained with nitrocefin but in
a slightly shorter time for S. aureus.
3.2. Nitroreductases
Using fluorogenic synthetic substrates, nitroreductase has been
detected with all microorganisms tested by James et al. (2001). The
sensitivity was stronger with the methyl 7-nitrocoumarin-3-carbox-
ylate than with the other 7-nitrocoumarins.
3.3. Luciferases
While the use of synthetic luminogenic substrates is limited with
intact cells due to reduced penetration of the exogenous substrate, the
emission of bioluminescence from substrate produced internally by
genetically modified bacteria is a powerful tool for the study of
pathogen behaviour in intact animals. The luxABCDE operon encodes
simultaneously the luciferase enzyme and the complex synthesizing
its fatty aldehyde substrate. Thanks to the very low level of the
background bioluminescence, the luxABCDE operon has been used
successfully as a highly sensitive reporter for the assessment of
L. monocytogenes infection of mice (Bron et al., 2006).
Table 2 summarises the enzymatic activities and families of syn-
thetic enzymatic substrates used in microbiological applications.
4. Enzymatic substrates in identification (ID) systems
Initially, tests used for microbial characterization to detect key
enzymes, e.g., urease and decarboxylases, were based on pH changes
after hydrolysis of the active substrate. Seidman and Link (1950) first
synthesized the self-indicating enzymatic substrate, o-nitrophenyl-β-
D-galactoside, for use in detection of β-galactosidase in E. coli
(Lederberg, 1950), and its broader application was described later
(Le Minor and Ben Hamida, 1962). Subsequently, a wealth of enzymatic
substrates became available that could be used for either direct or
indirect detection of enzymes through the release of various chromo-
phores or fluorophores.
One of the earliest commercial products to employ the use of such
substrates for microbial ID was api ZYM. Introduced in the mid-1970s
but not associated with any specific database (Monget, 1975), this
research tool gained widespread use in microbial characterization,
e.g., of Gram-negative anaerobes (Tharagonnet et al., 1977), Strepto-
coccaceae (Waitkins et al., 1980), yeasts (Casal and Linares, 1983),
Enterobacter spp. (Muytjens et al., 1984), Gram-negative veterinary
pathogens (Groom et al., 1986), etc., and led to the development of ID
products targeting specific microbial groups.
With few exceptions, commercial ID products incorporated the use
of enzymatic substrates with other tests (e.g., carbon substrates,
nitrogen substrates, growth in the presence of inhibitors). Enzyme
tests enabled differentiation of otherwise very close species pairs and
allowed more rapid results than tests requiring extended periods of
growth, e.g., utilization of carbohydrates.
 S. Orenga et al. / Journal of Microbiological Methods 79 (2009) 139–155 145

Table 2
Summary of targeted enzymatic activities, dyes and synthetic enzymatic substrates according to microbiological applications.

Enzyme Dye/substrate Microorganism Sample

Enumeration and isolation Glycosidase α-Galactosidase Indoxyl Salmonella C–Fa

plate medium Indoxyl E. coli O157:H7 C–F
β-Galactosidase Indoxyl Escherichia coli C
Indoxyl E. coli O157:H7 C–F
Indoxyl Coliform, F–W
Indoxyl Staphylococcus saprophyticus C
Alizarin VREb C
α-Glucosidase Indoxyl Cronobacter sakazakii F
Indoxyl Staphylococcus aureus C–F
Indoxyl MRSA C
Indoxyl VRE C
β-Glucosidase Indoxyl KESC C
Indoxyl ESBL producing enterobacteria C
Indoxyl Vibrio C–F
Indoxyl Enterococci C–W
Indoxyl VRE C
Indoxyl Listeria spp F
Indoxyl Candida kefyr, Candida lusitaniae, C
Candida tropicalis
Cellobiosidase Indoxyl Cronobacter sakazakii F
β-Glucuronidase 4-MU, Indoxyl E. coli C–F–W
Indoxyl ESBL producing E. coli C
Indoxyl Streptococcus agalactiae C
β-Ribofuranosidase Indoxyl, 3′,4′-Dihydroxyflavone Yersinia enterocolitica C
Hexosaminidase Indoxyl, VLPA Candida albicans, Candida dubliniensis C
Esterase Lipase Indoxyl Salmonella C–F
PI-PLC Indoxyl Listeria monocytogenes F
PC-PLC Indoxyl Bacillus F
Phosphatase Indoxyl S. aureus C–F
Indoxyl S. agalactiae C
4-MU, Indoxyl Clostridium perfringens F
Indoxyl Candida spp. C
Arylamidase/peptidase β-Alanyl arylamidase 7-Amido-1-pentyl-phenoxazin-3-one Pseudomonas aeruginosa C
Miscellaneous Deoxyribonuclease Indoxyl S. aureus F
Spot test Esterase Lipase 4-MU Salmonella C–F
Arylamidase/peptidase L-Alanyl arylamidase AMC, p-Nitroanilide Gram+/Gram− F
Miscellaneous β-Lactamase Nitrocefin, PADAC, CENTA, Cefesone β-lactamase producing bacteria C
Most probable number Glycosidase β-Glucuronidase 4-MU E. coli F
β-Galactosidase Nitrophenol, 4-MU Coliform F
Cytometry Glycosidase β-Glucuronidase Fluorescein E. coli W
β-Galactosidase Fluorescein Coliform W

Enzyme Dye Microorganism

Multi-test identification device Glycosidase Nitrophenol Aerobic Gram-negative bacteria

(detailed enzymatic activities: see Table 3) Naphthol Aerobic Gram-positive bacteria
Indoxyl Coryneforms
4-MU Yeasts
Resorufin Anaerobes
Fastidious Gram-negative
Esterase Nitrophenol Aerobic Gram-negative bacteria
Naphthol Aerobic Gram-positive bacteria
Indoxyl Coryneforms
4-MU Yeasts
Anaerobes
Fastidious Gram-negative
Arylamidase/peptidase p-Nitroanilide Aerobic Gram-negative bacteria
Naphthylamine Aerobic Gram-positive bacteria
AMC Coryneforms
DCAP Yeasts
Anaerobes
Fastidious Gram-negative
a
C, clinical; F, food; W, water.
b
VRE, vancomycin-resistant enterococci; MRSA, methicillin-resistant Staphylococcus aureus; ESBL, extended spectrum ß-lactamase; KESC, Klebsiella, Enterobacter, Serratia,
Citrobacter.

The late 1970s brought several manual products into the commercial
arena, while automated products were introduced in the 1980s. In the
1990s and over the last decade, technologic advances (higher levels of
automation, more sophisticated electronics, e.g., optical assemblies, and
availability of more enzyme substrates) allowed for more rapid and
accurate ID methods. Table 3 shows most of the enzymatic substrates
used in different commercial ID products.
Enzymatic tests became a cornerstone of commercial ID systems,
first applied to manual kits with later evolution to automated systems.
Enzymatic tests enabled more rapid identification with incubation
times as short as 2 h. Although these tests are significantly more rapid
than the more conventional growth-based tests, they may require a
higher inoculum density (e.g., with anaerobes and coryneforms),
which may require additional subculture.
146 S. Orenga et al. / Journal of Microbiological Methods 79 (2009) 139–155

Table 3 Table 3 (continued)

Enzymatic tests and applications in commercial ID products. Enzyme Application
Enzyme Application GNa GP YS AC FN
GNa GP YS AC FN Miscellaneous
Glycosidases Pyrazinamidase
N-acetyl-β-galactosaminidase Xb X X Tryptophan deaminase X X
N-acetyl-β-glucosaminidase X X X X Urease X X X X X
α-Amylase X a
GN, non-fastidious Gram-negative; GP, aerobic Gram-positive except coryneforms;
α-Arabinofuranosidase X YS, yeasts; AC, anaerobes/coryneforms; FN, fastidious Gram-negative.
α-Arabinosidase X X X b
X, used in corresponding identification reagent.
β-Cellobiosidase X X
β-Chitobiosidase X X
α-Fucosidase X The differential capabilities afforded by enzymatic tests have
β-Fucosidase X X enabled development of highly accurate ID methods surpassing those
α-Galactosidase X X X X available previously with only carbon and nitrogen substrate utilization
β-Galactosidase X X X X X
tests. Additionally, an increased use of polyphasic, i.e., phenotypic,
α-Glucosidase X X X X X
β-Glucosidase or esculin X X X X genotypic, and chemotaxonomic, characterization (Vandamme et al.,
hydrolysis 1996) has resulted in better-characterized culture collections and
β-Glucuronidase X X X X stronger knowledge bases also reinforcing the development of highly
α-Maltosidase X accurate phenotypic ID products.
β-Maltosidase X
α-Mannosidase X X
Currently, there are many commercial ID products with essentially
β-Mannosidase X X X equivalent performances and other features, e.g., workflow, cost, etc.,
β-Xylosidase X X X are key for their selection in various laboratories. Commercial ID
product evaluations typically show a wide range of accuracy most
Arylamidases
often due to differences in study design.
Alanyl arylamidase X X
β-Alanyl arylamidase X Influential factors of the study design include species and strain
Alanyl-alanine arylamidase X selection, comparative method(s), database version, etc. It is therefore
Alanyl-phenylalanyl-proline X X X X prudent to focus on several studies rather than a single one. While it is
arylamidase impractical to review all available products, the tables below focus on
Arginine arylamidase X X X X
some of the more widely used products with enzymatic substrates and
Arginyl-arginine arylamidase X
Aspartic acid arylamidase X X X their respective performances. It is noteworthy that some recent
Citrulline arylamidase X evaluations (e.g., Heikens et al., 2005, Layer et al., 2006, Sanguinetti
α-Glutamic acid arylamidase X X et al., 2007, Delmas et al., 2008) compare ID products to molecular
γ-Glutamyl transferase X X X
methods while older evaluations compare ID products to conventional
Glutamyl-glutamic arylamidase X
Glutamyl-glycyl-arginine X methods (e.g., Ruoff and Kunz, 1983, Facklam et al., 1985, O'Hara and
arylamidase Miller, 1992) or to other commercial products (e.g., Endimiani et al.,
Glycine arylamidase X X X X 2002, Stefaniuk et al., 2003, Donay et al., 2004). One cautionary note is
Glycyl-arginine arylamidase X that comparison of one commercial product to another commercial
Glycyl-glycine arylamidase X X X
product may compound an inherent error rate present in the com-
Glycyl-proline arylamidase X
Glycyl-tryptophan arylamidase X parator method. These and other factors can account for wide
Histidine arylamidase X X X differences in observed performances as shown in Tables 4–8.
Hydroxyproline arylamidase X
Isoleucine arylamidase X
5. Application of enzyme substrates in culture media
Leucine arylamidase X X X X
Leucyl-glycine arylamidase X
for microbiology
Lysine arylamidase X X X X
Lysyl-alanine arylamidase X Over the last 20 years, there has been a rapid expansion in the
Methionine arylamidase X X development and commercial availability of chromogenic agar media
Phenylalanine arylamidase X X X
for the detection of pathogenic bacteria and yeasts (Perry and Freydière,
Proline arylamidase X X X X X
Pyrrolidonyl arylamidase X X X X 2007). Such culture media typically contain multiple substrates that
Serine arylamidase X allow bacteria to form coloured colonies based on their enzymatic
Seryl-tyrosine arylamidase X activity. This facilitates the differentiation of species within polymicro-
Tryptophan arylamidase X
bial cultures and the targeting of pathogens with high specificity. When
Tyrosine arylamidase X X X X X
specific pathogens are targeted, selective agents such as antibiotics are
Esterases employed to limit the number of species able to grow.
Acetate esterase X Most media rely upon the inclusion of indoxylic substrates in order
Lipase X X to generate colonies with contrasting colours. For example, the release
Phosphatase X X X X X
of green and red chromogens from two distinct substrates can result
Phospholipase X
in the formation of green, red or purple colonies depending on
Miscellaneous whether one or both enzyme activities are present. Pathogens may
Arginine dihydrolase X X X therefore be differentiated from commensal bacteria by their
Gelatinase X X
possession of either one or both enzymes.
Hippurase X X
Glutamic acid decarboxylase X
Indoxylic glycosidase substrates are employed in the majority of
Indole formation (tryptophanase) X X X chromogenic media used in microbiology laboratories including media
Lysine decarboxylase X for the detection of urinary tract pathogens, Salmonella spp., E. coli
Nitrate reductase X X X X O157:H7, Cronobacter sakazakii, Vibrio, S. aureus, enterococci, group B
Ornithine decarboxylase X X X
streptococci, Listeria and yeasts. The glycosidases targeted are largely
Phenylalanine deaminase X
confined to a narrow range, which includes α- or β-galactosidase, α-
 S. Orenga et al. / Journal of Microbiological Methods 79 (2009) 139–155 147

Table 4 Table 5
Products for Enterobacteriaceae (E) and/or non-Enterobacteriaceae (NE). Products for Micrococcaceae and/or Streptococcaceae.

No. Type of % Reference No. Type of taxa % Reference

isolates taxa tested Correct isolates tested Correct

Manual products Manual products

api 20E 245 E 99.2 Hayek and Willis, 1976 api Staph 120 CNSa 88.3 Giger et al., 1984
258 NE 99.0 Appelbaum et al., 1984 212 Staphylococci 86.8 Baldellon and Mégraud, 1985
512 E/NE 98.6 Robinson et al., 1995 200 CNS 85.5 De Paulis et al., 2003
505 E 89.1 Wauters et al., 1995 58 CNS 87.9 Heikens et al., 2005
Crystal E/NF 266 E/NE 94.0 Holmes et al., 1994 api 20 Strep 209 Streptococci 85.2 Appelbaum et al., 1984
512 E/NE 95.5 Robinson et al., 1995 77 Viridans 90.0 Etienne et al., 1984
706 E/NE 88.7 Wauters et al., 1995 251 Streptococci 86.1 Facklam et al., 1984
626 E/NE 87.9 O'Hara et al., 1997 119 Viridans 92.0 Ruoff and Kunz, 1983
api 20 NE 262 NE 91.6 Appelbaum and Leathers, 1984 199 Streptococci 88.4 Watts, 1989
431 NE 97.0 von Graevenitz and Crystal GP 191 All GPC 90.0 von Baum et al., 1998
Zollinger-Iten, 1985 124 Enterococci 82.0 Jackson et al., 2004
229 NE 94.8 Martin et al., 1986 120 CNS 67.5 Kim et al., 2008
123 NE 95.1 Palmieri et al., 1988 ID 32 Staph 440 CNS 95.2 Ieven et al., 1995
201 NE 74.6 Wauters et al., 1995 200 CNS 79.5 Renneberg et al., 1995
ID 32 E 545 E/NE 92.8 Monnet et al., 1994 137 CNS 91.2 Edwards et al., 2001
524 E/NE 77.3 O'Hara and Miller, 1999 rapid ID 32 433 Streptococci and 95.3 Freney et al., 1992
RapID onE 379 E/NE 96.8 Kitch et al., 1994 Strep 156 related Viridans 87.2 Kikuchi et al., 1995
125 E 92.0 Lee et al., 1994 122 Streptococci/enterococci 93.0 Gorm Jensen et al., 1999
RapID NF Plus 345 NE 96.2 Kitch et al., 1992 124 Enterococci 85.0 Jackson et al., 2004
rapid ID 32E 414 E 95.4 Freney et al., 1991b RapID STR 266 Streptococci 97.0 Appelbaum et al., 1986
247 Streptococci 93.9 You and Facklam, 1986
Automated products
MicroScan Rapid 383 E/NE 95.3 Pfaller et al., 1991 Automated products
Neg Combo 3 467 E/NE 91.9 O'Hara and Miller, 1992 MicroScan 283 CNS 97.5 Weinstein et al., 1998
252 E/NE 96.4 O'Hara et al., 1993 Pos ID2 202 Enterococci 81.2 Iwen et al., 1999
MicroScan Rapid 652 E/NE 94.0 Bascomb et al., 1997 104 Staphylococci 94.2 Sáa et al., 1999
Neg ID3 511 E/NE 88.8 O'Hara and Miller, 2000 MicroScan 239 Staphylococci 95.4 Stoakes et al., 1992
134 NE 93.3 O'Hara and Miller, 2002 Rapid Pos 233 CNS 76.0 Grant et al., 1994
Phoenix NID 136 E/NE 95.6 Endimiani et al., 2002 285 CNS 88.7 Weinstein et al., 1998
174 E/NE 93.7 Stefaniuk et al., 2003 Phoenix PID 118 All GPC 89.8 Donay et al., 2004
187 E/NE 93.0 Donay et al., 2004 58 CNS 62.1 Heikens et al., 2005
564 E/NE 89.4 O'Hara, 2006 410 Staphylococci/enterococci 99.3 Carroll et al., 2006a
251 E 94.4 Carroll et al., 2006b 110 CNS 80.0 Layer et al., 2006
VITEK GNI+ 619 E/NE 87.6 O'Hara et al., 1997 200 Streptococci/enterococci 92.0 Brigante et al., 2006
304 E/NE 94.4 Bourbeau and Heiter, 1998 VITEK GPI 507 Streptococci 87.8 Facklam et al., 1985
299 NE 92.3 Sung et al., 2000 495 CNS 85.9 Bannerman et al., 1993
454 E 88.8 O'Hara and Miller, 2003 374 Enterococci 91.4 Willey et al., 1993
VITEK 2 GN 655 E/NE 99.4 Funke and Funke-Kissling, 2004 VITEK 2 GP 364 All GPC 94.5 Funke and Funke-Kissling, 2005
331 E/NE 97.0 Wallet et al., 2005 249 All GPC 94.4 Wallet et al., 2005
426 E/NE 99.5 Renaud et al., 2005 121 Enterococci 100.0 Abele-Horn et al., 2006
416 E/NE 97.1 Schreckenberger et al., 2005 110 CNS 90.9 Layer et al., 2006
168 Staphylococci 87.5 Delmas et al., 2008
120 CNS 95.0 Kim et al., 2008
a
CNS, coagulase negative staphylococci; GPC, Gram-positive cocci.

or β-glucosidase, β-glucuronidase and β-N-acetyl-hexosaminidase

activity. Glycosidase substrates may be combined with other sub-
strates for the detection of phosphatase or esterase activity in order to
target specific pathogens.

substitute the β-glucuronidase substrate for a β-galactosidase substrate.

5.1. Chromogenic media for clinical microbiology
5.1.1. Urinary tract pathogens
Infection of the urinary tract is usually diagnosed by semi-quantitative
culture of urine on agar media. Only a limited number of species are
commonly found in urine and these can be identified, or at least
differentiated from other species, by the inclusion of chromogenic
substrates. β-glucuronidase is expressed by around 94% of clinical isolates
of E. coli (the most common urinary tract pathogen) and is generally not
produced by other Gram-negative bacteria present in urine samples
(Kilian and Bülow, 1979). CPS ID 2 medium combines complementary
indoxylic substrates for β-glucuronidase and β-glucosidase to differen-
tiate E. coli and highlight β-glucosidase producers such as Klebsiella, En-
terobacter, Serratia and enterococci. Tryptophan is also included to
highlight species of the Proteeae (e.g., Proteus mirabilis) as brown colonies
due to their deaminase activity (Mazoyer et al., 1995).
An alternative approach employed in several media such as Uriselect
4, Oxoid Chromogenic UTI medium and CHROMagar Orientation is to
This allows detection of E. coli with higher sensitivity (99%) but with a
slightly lower specificity due to the occasional isolation of Citrobacter
freundii, which also produces β-galactosidase but often fails to express
β-glucosidase (Fallon et al., 2002, Perry et al., 2007). Both approaches
are effective for the isolation and differentiation of bacteria isolated from
urine samples (Carricajo et al., 1999).
5.1.2. Salmonella
In the diagnosis of gastroenteritis, stool samples are cultured onto
agar media to determine the presence of Salmonella spp. This requires
the use of sophisticated media in order to differentiate Salmonella spp.
from the large number of other species often present in the sample.
Chromogenic media for Salmonella use biochemical characteristics
such as acid formation from carbohydrates or the presence of C8
esterase for detection of Salmonella strains. Rambach agar and SM ID
were the first media of this type. Rambach agar employs X-Gal for β-D-
galactosidase-positive coliforms, Salmonella strains give red colonies
because of their ability to produce acid from propylene glycol revealed
148 S. Orenga et al. / Journal of Microbiological Methods 79 (2009) 139–155

Table 6 Table 8
Products for yeasts. Products for Neisseria (N), HACEK (H) group, and Campylobacter (C).

No. % Reference No. Type of taxa % Correct Reference

isolates Correct isolates tested

Manual products Manual products

api Candida 619 95.8 Fricker-Hidalgo et al., 1996 RapID NH 240 N 97.9 Robinson and Oberhofer, 1983
198 91.4 Bernal et al., 1998 187 H 89.8 Doern and Chapin, 1984
159 91.8 Campbell et al., 1999 299 H biotyping 92.0 Doern and Chapin, 1987
202 97.0 Paugam et al., 1999 90 N 94.4 Philip and Garton, 1985
RapID Yeast Plus 300 99.3 Kitch et al., 1996 380 H biotyping 51.6 Munson and Doern, 2007
447 97.6 Espinel-Ingroff et al., 1998 api Campy 100 C 92.0 Huysmans et al., 1995
133 94.0 Heelan et al., 1998 62 C 79.0 Reina et al., 1995
201 96.0 Wadlin et al., 1999 87 C 94.3 Shih, 2000
225 87.1 Smith et al., 1999 api NH 305 N/H 99.7 Barbé et al., 1994
750 95.5 Sanguinetti et al., 2007 380 H biotyping 97.6 Munson and Doern, 2007
VITEK NHI 480 N/H 95.2 Janda et al., 1987
Automated products 380 H biotyping 95.0 Munson and Doern, 2007
MicroScan Rapid 401 91.8 Land et al., 1991
Yeast 357 96.6 St.-Germain and Beauchesne, 1991 Automated products
150 67.0 Riddle et al., 1994 MicroScan HN 423 N/H 94.6 Janda et al., 1989
VITEK YBC 1106 94.3 Land et al., 1984 VITEK 2 NH 188 N/H/C 96.8 Valenza et al., 2007
221 83.3 Pfaller et al., 1988 371 N/H/C 96.5 Rennie et al., 2008b
150 85.0 Riddle et al., 1994
398 97.2 el-Zaatari et al., 1990
409 89.7 Fenn et al., 1994
VITEK 2 YST 97 99.0 Aubertine et al., 2006
623 98.6 Hata et al., 2007 et al., 1999). An alternative means of detecting Salmonella is exploited
750 98.4 Sanguinetti et al., 2007 in ABC medium, which utilizes an indoxylic α-galactoside (hydrolysed
136 94.1 Valenza et al., 2008 by Salmonella) in combination with 3,4-cyclohexenoesculetin-β-D-
galactoside (Perry et al., 1999). In order to improve the selectivity of
media for Salmonella, ‘suicide substrates’ such as alafosfalin have been
by neutral red as the pH indicator. SM ID also incorporates a β-D- exploited (Perry et al., 2002). Such substrates release toxic molecules
galactosidase substrate and glucuronic acid, which is metabolised by when hydrolysed so that commensal bacteria may be selectively
Salmonella spp. In most of the second generation of chromogenic inhibited on the basis of their ability to accumulate and hydrolyse
media, Salmonella is typically targeted by utilizing a substrate for C8 them. E. coli O157:H7 may also be recovered from stool samples
esterase activity. Few other species of Enterobacteriaceae produce C8 using specific chromogenic media designed for food samples (see
esterase and these may be differentiated by incorporation of a below).
substrate for β-galactosidase, or preferably β-glucosidase so that the
detection of lactose fermenting Salmonella is not precluded. Indoxylic 5.1.3. S. aureus
esters (e.g., octanoate or nonanoate) are normally used as substrates S. aureus is the most common cause of wound infection and diagnosis
but other chromogens have been utilized (Cooke et al., 1999; Gaillot is by culture of wound swabs onto culture media. Chromogenic media
have been developed for isolation and identification of this important
pathogen and such media typically employ an indoxylic substrate to
detect phosphatase activity. Phosphatase is produced by other species of
Table 7 staphylococci necessitating the use of complementary glycosidase
Products for anaerobes and coryneforms.
substrates, which are not hydrolysed by S. aureus, e.g., an indoxylic β-
No. % Reference glucoside. The use of a phosphatase substrate is exploited in several
isolates Correct commercially available chromogenic media including CHROMagar
Manual products Staph aureus (Gaillot et al., 2000). An alternative approach is to target
Crystal ANR 322 81.7 Cavallaro et al., 1997 α-glucosidase activity as employed in S. aureus ID (Perry et al., 2003).
ID 32 A 214 96.3 Kitch and Appelbaum, 1989
105 86.3 Grollier et al., 1992
102 96.1 Pattyn et al., 1993 5.1.4. Differentiation of yeasts
460 82.0 Downes et al., 1999 When different species of yeast are present in a polymicrobial
122 95.1 Sperner et al., 1999 culture, they are extremely difficult to distinguish from each other
RapID ANA II 300 87.0 Celig and Schreckenberger, 1991 using conventional media. Chromogenic media have been designed to
566 78.0 Marler et al., 1991
api Coryne 240 97.6
provide differentiation of species and identification of the most
Freney et al., 1991a
177 88.1 Gavin et al., 1992 important pathogenic yeast, Candida albicans. The latter is achieved
160 87.5 Soto et al., 1994 by the incorporation of a β-hexosaminidase substrate, which is
407 90.5 Funke et al., 1997 hydrolysed by C. albicans to generate coloured colonies. CHROMagar
178 91.0 Almuzara et al., 2006
Candida employs a further substrate for phosphatase activity allowing
50 72.0 Adderson et al., 2008
Crystal GP 50 76.0 Adderson et al., 2008 the specific identification of Candida tropicalis, which produces both
RapID CB Plus 86 80.2 Hudspeth et al., 1998 enzymes (Odds and Bernaerts, 1994).
378 80.9 Funke et al., 1998
50 52.0 Adderson et al., 2008 5.1.5. Group B Streptococcus
VITEK ANI 341 83.3 Schreckenberger et al., 1988
During antenatal screening many women are routinely screened
Automated products for the presence of Streptococcus agalactiae (group B Streptococcus) as
MicroScan Rapid 237 76.7 Stoakes et al., 1990 a vaginal commensal since this species may cause severe disease in
Anaerobe neonates. S. agalactiae may be differentiated from other vaginal flora
VITEK 2 ANC 365 95.1 Rennie et al., 2008a
by use of an indoxylic phosphate, which is hydrolysed to produce
251 92.0 Mory et al., 2009
coloured colonies. Phosphatase activity is commonly expressed by
 S. Orenga et al. / Journal of Microbiological Methods 79 (2009) 139–155
other species of the vaginal flora. It is therefore necessary to include
one or more complementary glycosidase substrates that are not
hydrolysed by S. agalactiae to ensure that other species do not
resemble S. agalactiae (Perry et al., 2006b; Tazi et al., 2009).
5.1.6. Antibiotic-resistant bacteria
It has become increasingly common to screen hospitalized patients
for the presence of antibiotic-resistant bacteria as part of infection-
control practices. This practice is well established with respect to
limiting the spread of methicillin-resistant S. aureus (MRSA). Patients
found to harbour MRSA (e.g., nasal colonization) may be isolated and
subjected to decontamination therapy in order to limit the spread of
this important pathogen. Chromogenic media have been adapted to
detect resistant bacteria by inclusion of additional selective agents, thus
ensuring that only resistant strains of the target species are allowed to
grow. For example, CHROMagar Staph aureus and S. aureus ID have
both been adapted for isolation of MRSA by incorporation of methicillin
or a surrogate antimicrobial such as cefoxitin (Flayhart et al., 2005;
Perry et al., 2004). CPS ID 3 medium and CHROMagar Orientation have
been adapted for detection of Enterobacteriaceae producing extended
spectrum β-lactamases (Glupczynski et al., 2007; Randall et al., 2009).
The screening of stool samples to detect colonization with
vancomycin-resistant enterococci is also advocated by some author-
ities and specific chromogenic media have been designed for this
purpose. Typically, a highly selective medium is used (including
vancomycin) and a single substrate is incorporated for detection of all
enterococci, such as esculin or an indoxylic β-glucoside. Alternatively,
chromID VRE employs a mixture of substrates for detection of α-
glucosidase and β-galactosidase to allow detection and differentiation
of the two most important species; Enterococcus faecalis and Entero-
coccus faecium (Ledeboer et al., 2007).
5.1.7. P. aeruginosa
Recently, a new chromogenic culture medium has been described for
the isolation and identification of P. aeruginosa. This is the first agar
medium to employ a chromogenic substrate for detection of amino-
peptidase activity. Among the bacteria encountered clinically, β-alanyl
aminopeptidase is largely restricted to P. aeruginosa and a few
other Gram-negative species and this activity is detected by inclusion
of β-alanyl pentylresorufamine, which is hydrolysed to generate a
purple colouration (Zaytsev et al., 2008). This medium has been
employed for the direct identification of P. aeruginosa from the sputa
of patients with cystic fibrosis (Laine et al., 2009).
5.2. Fluorogenic and chromogenic media in food and water microbiology
In recent years, a number of selective chromogenic plating media
for detection and enumeration of the most important bacteria in food
and water have been developed and marketed (Manafi, 2000).
5.2.1. E. coli and coliforms
The new generation of media uses β-D-glucuronidase as the
indicator for E. coli and β-D-galactosidase as the indicator for coliforms.
There are a wide range of media detecting the presence of coliforms
and E. coli using different chromogenic or fluorogenic enzyme sub-
strates that have been reviewed before (Manafi 1996, 2000). Based on
such substrates, specific devices have been designed and successfully
tested for enumeration of E. coli from water samples: Petrifilm E. coli/
coliform count plates, m-ColiBlue, Colilert-18 and Quanti-Tray 2000,
(Vail et al., 2003) or food samples (Tempo EC, Torlak et al., 2008).
Combining solid phase cytometry and separation of the growth/
enzyme induction phase from the enzyme activity measurement with
fluorescein-di-β-glucuronide or fluorescein-di-β-galactopyranoside,
it is possible to enumerate E. coli or coliforms from water samples in
less than 4 h (Nelis and Van Poucke, 2000, Van Poucke and Nelis,
149
2000a), which was not achievable by visual or CCD-camera fluores-
cence detection (Van Poucke and Nelis, 2000b).
5.2.2. E. coli O157:H7
Several chromogenic media have been applied to the detection
of E. coli O157:H7 from food samples. Most are based on similar
principles; relying on non-acidification from sorbitol and/or rham-
nose and lack of β-D-glucuronidase activity. A second chromogenic
substrate (e.g., for α- or β-D-galactosidase) may be used to highlight
the presence of E. coli O157:H7 among non-reactive background flora.
The performance of three chromogenic agars: Rainbow Agar O157,
CHROMagar O157 (pink colonies) and O157:H7 ID agar (green
colonies), with a large collection of verotoxigenic and non-toxigenic
E. coli strains have been tested (Bettelheim, 1998, 2005). Another
study compared Rainbow Agar O157, BCM O157:H7 and Fluorocult HC
with the conventional Sorbitol MacConkey and showed the clear
advantage of the chromogenic media for the isolation of E. coli O157:
H7 from raw ground beef and raw drinking milk (Manafi and
Kremsmaier, 2001). The behaviour of E. coli O157:H7 was studied
during the manufacture and ripening of raw goat milk lactic cheeses
using O157:H7 ID agar and CT-SMAC agar (Vernozy-Rozand et al.,
2005). In conclusion, O157:H7 ID agar proved to be well suited and
simplified many of the inherent problems associated with plate
confirmation of E. coli O157:H7 using Sorbitol MacConkey agar as the
subculture medium.
5.2.3. Salmonella
Chromogenic media for Salmonella detection in clinical samples are
also used for food samples. One of the rare studies in food microbiology
using chromogenic media was done by Schönenbrücher et al. (2008).
The draft ISO 6579:2002 was compared to the European gold standard
(DIN EN 12824:1998), including the three new chromogenic plating
media AES Salmonella Agar Plate (ASAP), Oxoid Salmonella Chromo-
genic Medium (OSCM) and Miller-Mallinson agar (MM). Only on MM
agar, did all of the 36 tested type strains produce typical colonies,
especially strains of Salmonella Senftenberg, Salmonella arizonae, Sal-
monella Dublin and Salmonella Derby.
5.2.4. Shigella spp.
Warren et al. (2005) described the evaluation of a new chromogenic
medium (CSPM) for detection of Shigella spp. in food microbiology.
Colony colour enhancements created by CSPM may ease differentiation
of Shigella colonies from those of closely related bacteria.
5.2.5. C. sakazakii
C. (Enterobacter) sakazakii is an occasional contaminant of powdered
infant formula that can cause rare but severe food-borne infections in
infants. C. sakazakii possesses α-glucosidase activity, and a number of
selective, chromogenic agars for C. sakazakii isolation based on this
enzyme have been developed (Iversen and Forsythe, 2007, Lehner et al.,
2006, Restaino et al., 2006). The substrate 5-bromo-4-chloro-3-indolyl-
α-D-glucopyranoside (X-α-Glc) is added to a basal medium to dif-
ferentiate C. sakazakii strains from other members of the Enterobacter-
iaceae. The enzyme α-glucosidase hydrolyses X-α-Glc giving blue
coloured colonies on DFI agar, ESIA agar, chromID Sakazakii or
Chromocult Enterobacter sakazakii (CES) (Cawthorn et al., 2008),
which are commercially available. A modification of DFI agar supple-
mented with glucose (mDFI) was described by Song et al. (2008) to
eliminate false positive results with Escherichia vulneris.
5.2.6. Y. enterocolitica
Weagant (2008) described an agar for detection of potentially
virulent Y. enterocolitica (YeCM). This medium contains cellobiose as
the fermentable sugar and a chromogenic substrate. Y. enterocolitica
biotype 1A and other related Yersinia formed colonies that were
150 S. Orenga et al. / Journal of Microbiological Methods 79 (2009) 139–155
purple/blue on YeCM while they formed typical red bulls-eye colonies
on Cefsulodin-Irgasan-Novobiocin agar.
5.2.7. Vibrio spp.
Thiosulphate-Citrate-Bile-Sucrose agar used in the laboratory is
not very selective and cannot differentiate between different Vibrio
species. There are new chromogenic media for Vibrio, which enable
pathogenic species such as Vibrio parahaemolyticus, Vibrio cholerae,
and Vibrio vulnificus to be differentiated on the basis of chromogenic
reactions. Chromochecker Vibrio agar for detection of V. vulnificus
(Nakashima et al., 2007), VVM (Cerdà-Cuéllar et al., 2000), Bio-
Chrome Vibrio medium (Su et al., 2005) and commercially available
media CHROMagar Vibrio (Hara-Kudo et al., 2001, Blanco-Abad et al.,
2009) and chromID Vibrio are new and further evaluation studies are
needed.
5.2.8. Enterococci
Substrates such as 5-bromo-4-chloro-3-indolyl-β-D-glucopyranoside
(X-Glu) and indoxyl-β-D-glucoside were already described for de-
tection of β-D-glucosidase activity (Manafi, 2000). Enterococci that are
β-glucosidase-positive produce an insoluble indigoid blue complex,
which diffuses into the surrounding medium, forming a blue halo around
the colony. Chromocult Enterococci broth and Readycult Enterococci
utilize the β-D-glucosidase reaction as an indicator using X-Glu. The
X-Glu is liberated and rapidly oxidized to dibromodichloroindigo, which
produces blue colour in Chromocult broth. Chromocult enterococci agar
uses a chromogenic mix in a selective agar; enterococci cleave
chromogenic substrates in this medium and show red coloured colonies
on the plate. Non-enterococci produce colourless, blue/violet or
turquoise colonies (Miranda et al., 2005).
5.2.9. C. perfringens
The identification of C. perfringens is possible using Fluorocult
TSCagar using a fluorogenic enzyme substrate. D-Cycloserine inhi-
bits the accompanying bacterial flora and causes the colonies to
remain smaller. C. perfringens colonies can be detected using 4-MU-
phosphate. Acid phosphatase is a highly specific indicator for C.
perfringens, which shows a light blue fluorescence on this medium.
The new chromoselect CP agar, is now available, which can be used
in water microbiology. C. perfringens colonies give green coloured
colonies, and other clostridia species give violet, or blue coloured
colonies (Sigma, Switzerland).
5.2.10. L. monocytogenes
Several studies have been performed using chromogenic media for
the detection of L. monocytogenes and are summarised by Reissbrodt
(2004). There are two groups of such media: the first, including Agar
Listeria according to Ottaviani and Agosti (ALOA), utilizes cleavage by
phosphatidylinositol-specific phospholipase C (PI-PLC) of L-α-phospha-
tidylinositol, forming a white precipitation zone around the colony,
combined with the chromogenic substrate 5-bromo-4-chloro-3-indoxyl-
β-D-glucopyranoside for detection of β-D-glucosidase, which occurs in
all Listeria spp. The typical colony morphology of Listeria spp. is reported
to be turquoise blue. Pathogenic Listeria colonies are additionally
surrounded by a translucent halo. The second group of media utilizes
5-bromo-4-chloro-3-indoxyl-myoinositol-1-phosphate, forming blue-
turquoise colonies of pathogenic Listeria spp.: L. monocytogenes and L.
ivanovii. A recent paper (Stessl et al., 2009) evaluated six chromogenic
media similar to ALOA, in addition testing the ability to facilitate growth
of stressed L. monocytogenes strains and mixed cultures with competitive
non-Listeria and enumeration of L. monocytogenes in food samples. They
found that the competitive flora of food samples is able to overgrow low
numbers of L. monocytogenes, especially in half-Fraser enrichment. This
might lead to the underestimation of L. monocytogenes-positive samples.
Examples of such media include CHROMagar Listeria agar, OCLA,
Compass L. mono, chromID OAA and Chromoplate Listeria.
5.2.11. B. cereus and Bacillus thuringiensis
Phosphatidylcholine-specific phospholipase C (PC-PLC), encoded
by the plc genes, is used as a marker for these two microorganisms.
Hydrolysis of 5-bromo-4-chloro-3-indoxyl-choline-phosphate yields
blue–green colonies, indicating the presence of PC-PLC activity
(Juergensmeyer et al., 2006). The Chromogenic Bacillus Cereus agar
contains 5-bromo-4-chloro-3-indolyl-β-D-glucopyranoside that is
cleaved by β-D-glucosidase and results in white colonies with a
blue–green centre (Fricker et al., 2008).
5.2.12. Bacillus anthracis
The chromogenic R&F Anthracis chromogenic agar (ChrA) is
described and evaluated by Marston et al. (2008). Thirteen of the 16
B. anthracis tested strains produced the expected morphology on the
ChrA medium.
6. Conclusion
Synthetic enzymatic substrates have long been useful for both
fundamental microbiology (Jacob and Monod, 1961) and daily analysis
of clinical, food and environmental samples. Despite the development
of identification methods based on direct nucleic acid, fatty acid,
protein or antigen analysis, they remain powerful tools for detection,
enumeration and identification of microorganisms giving simulta-
neously descriptive and functional information. For the detection of
carbapenemase producing Enterobacteriaceae, they allow for a reduc-
tion in the use of expensive and complex phenotypic or molecular tests
to a limited number of presumptive positive isolates (CDC, 2009).
Finally, thanks to the continuous development of new molecules, they
also participate to highlight the microbial complexity, as illustrated by
the number of colony variants of P. aeruginosa isolated from sputum
samples of cystic fibrosis patients (Laine et al., 2009).
Disclosure statement
In the last three years, Arthur James has received financial support
for consultancy from bioMérieux. He also receives financial remuner-
ation from sales of identification devices and a chromogenic medium
supplied by bioMérieux.
In the last three years, John Perry has received financial support for
research or consultancy from suppliers of chromogenic culture media
including bioMérieux, Becton Dickinson and Bio-Rad. He also receives
financial remuneration from sales of a chromogenic medium supplied
by International Diagnostics Group (Lab M).
David Pincus and Sylvain Orenga have been employed by bioMérieux
for the last three years. Sylvain Orenga also received financial re-
muneration for patent applications and registrations.
Mammad Manafi has no potential conflict to declare.
Acknowledgment
The authors thank Daniel Monget for his broad and rigorous
involvement in the field of enzymatic substrates for microbiology
especially for the improvement of microbial identification. By the
results of his work as well as by direct training of some of us, he
contributed deeply to our knowledge. And even more, he allowed us
to meet and work together in a creative and friendly relationship. As
such, he indirectly inspired and contributed to this review.
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