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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
As the indigoid dyes formed upon intracellular hydrolysis of Ichibangase et al. (2004) designed a chemiluminescence assay for lipase
indoxyl based substrates remain inside cells, they can be used for using the lauric acid ester of 2-(4-hydroxyphenyl)-4,5-diphenylimidazole
single cell characterization (Poulsen et al., 1997). (HDI) as a substrate. The generated HDI acts as an enhancer of the
Bainbridge et al. (1991) tried to develop substrates with higher chemiluminescence reaction of luminol–hydrogen peroxide–horseradish
water solubility and lower cost than their indoxyl equivalent. However, peroxidase. While this approach was successful for the assay of lipase
their proposed 2-(2-(4-(β-D-galactopyranosyloxy)-3-methoxyphenyl)- from Candida cylindracea and from porcine pancreas, it seems limited to
vinyl)-3-methylbenzothiazolium toluene-4-sulphonate had to be used assay free enzyme rather than analyse whole living cells. To increase the
with either filter paper or nitrocellulose membranes and in a limited pH sensitivity of the detection of β-galactosidase activity from coliform cells,
range. The application on colonies of a filter paper impregnated with the Van Poucke and Nelis (1995 introduced a permeabilization step with
enzymatic substrate was also required for the lipase substrates based on polymyxin B between the induction of cellular enzymatic activity and the
5-(4-hydroxy-3,5-dimethoxyphenylmethylene)-2-thioxothiazoline-3- measurement of luminescence produced in the presence of a chloro
acetic acid (Miles et al., 1992). Cooke et al. (2002) overcame this derivative of 3-{4-methoxyspiro [1,2-dioxetane-3,2′-tricyclo(3.3.1.13,7)
limitation in a chromogenic medium for Candida with a closely related decan]-yl}phenyl-β-D-galactopyranoside. The phosphatidylinositol-
substrate targeting hexosaminidase activity: 4-(2-[4-(2-acetamido-2- phospholipase C activity from culture supernatant of Bacillus cereus
deoxy-β-D-glucopyranosyloxy)-3-methoxyphenyl]-vinyl)-1-(propan- was detected with a luminogenic substrate based on an adamantane–
3-yl-oate)-quinolium bromide (VLPA-GlcNAc). On their medium, more dioxetane–phenyl group (Ryan et al., 1993).
yeast species hydrolysed the hexosaminidase substrate to produce pink The bioluminogenic substrate D-luciferin-o-β-galactopyranoside is
to red colonies than on chromogenic media incorporating 5-bromo-4- cleaved by β-galactosidase to release luciferin. This luciferin serves as
chloro-3-indoxyl-N-acetyl-β-D-glucosaminide. a substrate for luciferase (Geiger et al., 1992). For microorganism
To overcome some limitations of indoxyl based substrates and analysis, it is combined with a detergent that lyses cells to release the
especially the costs and the requirement of aerobic incubation, James β-galactosidase present (de Almeida et al., 2008).
et al. (2000a) synthesized p-naphtholbenzein-β-D-galactopyranoside
(pNB-gal). When incorporated in agar media, β-galactosidase pro- 2.4. Secondary reaction based substrates
ducing colonies were pink. For similar reasons, James et al. (2000b)
synthesized alizarin-β-D-galactoside (Aliz-gal). Upon hydrolysis, the In some cases, the detection of enzymatic activity still relies on
generated alizarin chelates with ferrous or aluminium ions forming detection of the natural metabolic product. This is the case for
brightly coloured complexes. tryptophanase that metabolises tryptophan to generate indole. The
When tested on strains of Enterobacteriaceae in the presence indole may be detected by reaction with various aldehydes such as 4-
of isopropyl-β-D-thiogalactopyranoside (IPTG), the sensitivity of dimethylaminobenzaldehyde (DMABA), 4-dimethylaminocinnamalde-
pNB-gal was equivalent to that of X-β-D-galactopyranoside (X-gal) hyde (DMACA), or 2-methoxy-4-dimethylaminobenzaldehyde (James
except for some strains of Serratia and Yersinia enterocolitica (James et al., 1986).
et al., 2000a). With Aliz-gal, the sensitivity was slightly higher than Due to low sensitivity of L-alanyl-p-nitroanilide for detecting the
with X-gal for some strains of Serratia and Yersinia (James et al., D-alanyl-D-alanyl dipeptidase VanX from vancomycin-resistant entero-
2000b). cocci, Anissimova et al. (2003) developed an assay based on D-alanyl-α-
To detect β-glucuronidase activity of colonies of Escherichia coli, (R)-phenylthioglycine. The released α-phenylthioglycine was detected
James and Yeoman (1988) used 8-hydroxyquinoline-β-D-glucuronide. upon reaction with 5,5′-dithio-bis-(2-nitrobenzoic acid) (Ellman's re-
The released 8-hydroxyquinoline chelates with ferrous ions present agent) and could be continuously monitored spectrophotometrically.
in the medium generating an intense black pigmentation, but the
sensitivity was lower than that observed with 4-MU-β-D-glucuronide. 2.5. Suicide substrates
Black pigmentation is also obtained with the use of substrates based on
3,4-cyclohexenoesculetin (James et al., 1996, 1997), 3′,4′-dihydroxy- As in oncology, microbiologists use some enzymatic activities to
flavone (Butterworth et al., 2004) and 3-hydroxyflavone (Perry et al., inhibit specifically bacteria expressing those activities. While the
2006a). In those cases, the sensitivity was similar to that obtained with toxicity of the substrate is very low, one of the products of the
indoxyl substrates for similar enzymatic activities. metabolism is highly toxic. It is useful especially with intracellular
Aminophenyl-acridine based substrates can be used for peptidase enzymatic activities such as the β-galactosidase of E. coli (Park et al.,
detection (James et al., 2007). The 9-(4′-aminophenyl)-acridines are pale 1976). The application of this technology has been further extended
yellow, but when protonated either as an acridinium salt or by the application of phosphonopeptides (Perry et al., 2002).
upon addition of acetic acid after incubation, they turn red. Anderson
et al. (2008) used such novel chromogenic peptidase substrates based on 3. Microbial enzymatic activities
9-(4′-aminophenyl)-10-methylacridinium salts for direct detection of
microbial peptidase in colonies on agar plate media. Some of them appear 3.1. Hydrolases
useful for differentiation among enterobacteria species. Chromogenic
peptidase substrates can also be based on derivatives of 7-amino- Most of the synthetic enzymatic substrates used in microbiology are
phenoxazin-3-one (resorufamine, Zaytsev et al., 2008). The 1-pentyl substrates targeting hydrolases (Fig. 2). In particular, a wide range of
substituted derivative (pentylresorufamine, PRF) localises in the bacterial glycosidases, has been exploited as enzymatic targets (Manafi et al.,
cells, producing purple colonies on agar plate media (Fig. 1), but the L- 1991). Substrates for β-ribofuranosidase appear useful for differentia-
alanyl-PRF was inhibitory to Gram-positive bacteria. tion between Y. enterocolitica, which does not hydrolyse these sub-
strates, and most other enterobacteria, which are positive (Butterworth
2.3. Luminogenic substrates et al., 2004). A complex glycoside, 5-bromo-4-chloro-3-indoxyl-5′-
acetamido-3′,5′-dideoxy-α-D-glycero-D-galacto-2-nonulopyranosido-
Luminescence enables detection with high sensitivity and wide nic acid, is hydrolysed by a neuraminidase from Clostridium perfringens
dynamic range. The free enzyme assay of β-D-galactosidase based on producing an insoluble indigoid dye upon aerobic oxidation (Fujii et al.,
the chemiluminogenic substrate o-aminophthalylhydrazide-β-D-galacto- 1993). However, as C. perfringens requires an anaerobic atmosphere, it is
side has a sensitivity 10 times higher than the fluorometric method based not adapted to direct detection of growing C. perfringens strains.
on 4-MU-β-D-galactoside and 100 times higher than the colorimetric Deoxyribonuclease activity is expressed by Staphylococcus aureus
method based on o-nitrophenyl-β-D-galactoside (Nakazono et al., 1992). 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.
143
144 S. Orenga et al. / Journal of Microbiological Methods 79 (2009) 139–155
3.2. Nitroreductases
3.3. Luciferases
Table 2
Summary of targeted enzymatic activities, dyes and synthetic enzymatic substrates according to microbiological applications.
The late 1970s brought several manual products into the commercial Enzymatic tests became a cornerstone of commercial ID systems,
arena, while automated products were introduced in the 1980s. In the first applied to manual kits with later evolution to automated systems.
1990s and over the last decade, technologic advances (higher levels of Enzymatic tests enabled more rapid identification with incubation
automation, more sophisticated electronics, e.g., optical assemblies, and times as short as 2 h. Although these tests are significantly more rapid
availability of more enzyme substrates) allowed for more rapid and than the more conventional growth-based tests, they may require a
accurate ID methods. Table 3 shows most of the enzymatic substrates higher inoculum density (e.g., with anaerobes and coryneforms),
used in different commercial ID products. which may require additional subculture.
146 S. Orenga et al. / Journal of Microbiological Methods 79 (2009) 139–155
Table 4 Table 5
Products for Enterobacteriaceae (E) and/or non-Enterobacteriaceae (NE). Products for Micrococcaceae and/or Streptococcaceae.
Table 6 Table 8
Products for yeasts. Products for Neisseria (N), HACEK (H) group, and Campylobacter (C).
other species of the vaginal flora. It is therefore necessary to include 2000a), which was not achievable by visual or CCD-camera fluores-
one or more complementary glycosidase substrates that are not cence detection (Van Poucke and Nelis, 2000b).
hydrolysed by S. agalactiae to ensure that other species do not
resemble S. agalactiae (Perry et al., 2006b; Tazi et al., 2009). 5.2.2. E. coli O157:H7
Several chromogenic media have been applied to the detection
5.1.6. Antibiotic-resistant bacteria of E. coli O157:H7 from food samples. Most are based on similar
It has become increasingly common to screen hospitalized patients principles; relying on non-acidification from sorbitol and/or rham-
for the presence of antibiotic-resistant bacteria as part of infection- nose and lack of β-D-glucuronidase activity. A second chromogenic
control practices. This practice is well established with respect to substrate (e.g., for α- or β-D-galactosidase) may be used to highlight
limiting the spread of methicillin-resistant S. aureus (MRSA). Patients the presence of E. coli O157:H7 among non-reactive background flora.
found to harbour MRSA (e.g., nasal colonization) may be isolated and The performance of three chromogenic agars: Rainbow Agar O157,
subjected to decontamination therapy in order to limit the spread of CHROMagar O157 (pink colonies) and O157:H7 ID agar (green
this important pathogen. Chromogenic media have been adapted to colonies), with a large collection of verotoxigenic and non-toxigenic
detect resistant bacteria by inclusion of additional selective agents, thus E. coli strains have been tested (Bettelheim, 1998, 2005). Another
ensuring that only resistant strains of the target species are allowed to study compared Rainbow Agar O157, BCM O157:H7 and Fluorocult HC
grow. For example, CHROMagar Staph aureus and S. aureus ID have with the conventional Sorbitol MacConkey and showed the clear
both been adapted for isolation of MRSA by incorporation of methicillin advantage of the chromogenic media for the isolation of E. coli O157:
or a surrogate antimicrobial such as cefoxitin (Flayhart et al., 2005; H7 from raw ground beef and raw drinking milk (Manafi and
Perry et al., 2004). CPS ID 3 medium and CHROMagar Orientation have Kremsmaier, 2001). The behaviour of E. coli O157:H7 was studied
been adapted for detection of Enterobacteriaceae producing extended during the manufacture and ripening of raw goat milk lactic cheeses
spectrum β-lactamases (Glupczynski et al., 2007; Randall et al., 2009). using O157:H7 ID agar and CT-SMAC agar (Vernozy-Rozand et al.,
The screening of stool samples to detect colonization with 2005). In conclusion, O157:H7 ID agar proved to be well suited and
vancomycin-resistant enterococci is also advocated by some author- simplified many of the inherent problems associated with plate
ities and specific chromogenic media have been designed for this confirmation of E. coli O157:H7 using Sorbitol MacConkey agar as the
purpose. Typically, a highly selective medium is used (including subculture medium.
vancomycin) and a single substrate is incorporated for detection of all
enterococci, such as esculin or an indoxylic β-glucoside. Alternatively, 5.2.3. Salmonella
chromID VRE employs a mixture of substrates for detection of α- Chromogenic media for Salmonella detection in clinical samples are
glucosidase and β-galactosidase to allow detection and differentiation also used for food samples. One of the rare studies in food microbiology
of the two most important species; Enterococcus faecalis and Entero- using chromogenic media was done by Schönenbrücher et al. (2008).
coccus faecium (Ledeboer et al., 2007). The draft ISO 6579:2002 was compared to the European gold standard
(DIN EN 12824:1998), including the three new chromogenic plating
5.1.7. P. aeruginosa media AES Salmonella Agar Plate (ASAP), Oxoid Salmonella Chromo-
Recently, a new chromogenic culture medium has been described for genic Medium (OSCM) and Miller-Mallinson agar (MM). Only on MM
the isolation and identification of P. aeruginosa. This is the first agar agar, did all of the 36 tested type strains produce typical colonies,
medium to employ a chromogenic substrate for detection of amino- especially strains of Salmonella Senftenberg, Salmonella arizonae, Sal-
peptidase activity. Among the bacteria encountered clinically, β-alanyl monella Dublin and Salmonella Derby.
aminopeptidase is largely restricted to P. aeruginosa and a few
other Gram-negative species and this activity is detected by inclusion 5.2.4. Shigella spp.
of β-alanyl pentylresorufamine, which is hydrolysed to generate a Warren et al. (2005) described the evaluation of a new chromogenic
purple colouration (Zaytsev et al., 2008). This medium has been medium (CSPM) for detection of Shigella spp. in food microbiology.
employed for the direct identification of P. aeruginosa from the sputa Colony colour enhancements created by CSPM may ease differentiation
of patients with cystic fibrosis (Laine et al., 2009). of Shigella colonies from those of closely related bacteria.
5.2. Fluorogenic and chromogenic media in food and water microbiology 5.2.5. C. sakazakii
C. (Enterobacter) sakazakii is an occasional contaminant of powdered
In recent years, a number of selective chromogenic plating media infant formula that can cause rare but severe food-borne infections in
for detection and enumeration of the most important bacteria in food infants. C. sakazakii possesses α-glucosidase activity, and a number of
and water have been developed and marketed (Manafi, 2000). selective, chromogenic agars for C. sakazakii isolation based on this
enzyme have been developed (Iversen and Forsythe, 2007, Lehner et al.,
5.2.1. E. coli and coliforms 2006, Restaino et al., 2006). The substrate 5-bromo-4-chloro-3-indolyl-
The new generation of media uses β-D-glucuronidase as the α-D-glucopyranoside (X-α-Glc) is added to a basal medium to dif-
indicator for E. coli and β-D-galactosidase as the indicator for coliforms. ferentiate C. sakazakii strains from other members of the Enterobacter-
There are a wide range of media detecting the presence of coliforms iaceae. The enzyme α-glucosidase hydrolyses X-α-Glc giving blue
and E. coli using different chromogenic or fluorogenic enzyme sub- coloured colonies on DFI agar, ESIA agar, chromID Sakazakii or
strates that have been reviewed before (Manafi 1996, 2000). Based on Chromocult Enterobacter sakazakii (CES) (Cawthorn et al., 2008),
such substrates, specific devices have been designed and successfully which are commercially available. A modification of DFI agar supple-
tested for enumeration of E. coli from water samples: Petrifilm E. coli/ mented with glucose (mDFI) was described by Song et al. (2008) to
coliform count plates, m-ColiBlue, Colilert-18 and Quanti-Tray 2000, eliminate false positive results with Escherichia vulneris.
(Vail et al., 2003) or food samples (Tempo EC, Torlak et al., 2008).
Combining solid phase cytometry and separation of the growth/ 5.2.6. Y. enterocolitica
enzyme induction phase from the enzyme activity measurement with Weagant (2008) described an agar for detection of potentially
fluorescein-di-β-glucuronide or fluorescein-di-β-galactopyranoside, virulent Y. enterocolitica (YeCM). This medium contains cellobiose as
it is possible to enumerate E. coli or coliforms from water samples in the fermentable sugar and a chromogenic substrate. Y. enterocolitica
less than 4 h (Nelis and Van Poucke, 2000, Van Poucke and Nelis, 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 5.2.11. B. cereus and Bacillus thuringiensis
on Cefsulodin-Irgasan-Novobiocin agar. Phosphatidylcholine-specific phospholipase C (PC-PLC), encoded
by the plc genes, is used as a marker for these two microorganisms.
5.2.7. Vibrio spp. Hydrolysis of 5-bromo-4-chloro-3-indoxyl-choline-phosphate yields
Thiosulphate-Citrate-Bile-Sucrose agar used in the laboratory is blue–green colonies, indicating the presence of PC-PLC activity
not very selective and cannot differentiate between different Vibrio (Juergensmeyer et al., 2006). The Chromogenic Bacillus Cereus agar
species. There are new chromogenic media for Vibrio, which enable contains 5-bromo-4-chloro-3-indolyl-β-D-glucopyranoside that is
pathogenic species such as Vibrio parahaemolyticus, Vibrio cholerae, cleaved by β-D-glucosidase and results in white colonies with a
and Vibrio vulnificus to be differentiated on the basis of chromogenic blue–green centre (Fricker et al., 2008).
reactions. Chromochecker Vibrio agar for detection of V. vulnificus
(Nakashima et al., 2007), VVM (Cerdà-Cuéllar et al., 2000), Bio- 5.2.12. Bacillus anthracis
Chrome Vibrio medium (Su et al., 2005) and commercially available The chromogenic R&F Anthracis chromogenic agar (ChrA) is
media CHROMagar Vibrio (Hara-Kudo et al., 2001, Blanco-Abad et al., described and evaluated by Marston et al. (2008). Thirteen of the 16
2009) and chromID Vibrio are new and further evaluation studies are B. anthracis tested strains produced the expected morphology on the
needed. ChrA medium.
5.2.8. Enterococci
6. Conclusion
Substrates such as 5-bromo-4-chloro-3-indolyl-β-D-glucopyranoside
(X-Glu) and indoxyl-β-D-glucoside were already described for de-
Synthetic enzymatic substrates have long been useful for both
tection of β-D-glucosidase activity (Manafi, 2000). Enterococci that are
fundamental microbiology (Jacob and Monod, 1961) and daily analysis
β-glucosidase-positive produce an insoluble indigoid blue complex,
of clinical, food and environmental samples. Despite the development
which diffuses into the surrounding medium, forming a blue halo around
of identification methods based on direct nucleic acid, fatty acid,
the colony. Chromocult Enterococci broth and Readycult Enterococci
protein or antigen analysis, they remain powerful tools for detection,
utilize the β-D-glucosidase reaction as an indicator using X-Glu. The
enumeration and identification of microorganisms giving simulta-
X-Glu is liberated and rapidly oxidized to dibromodichloroindigo, which
neously descriptive and functional information. For the detection of
produces blue colour in Chromocult broth. Chromocult enterococci agar
carbapenemase producing Enterobacteriaceae, they allow for a reduc-
uses a chromogenic mix in a selective agar; enterococci cleave
tion in the use of expensive and complex phenotypic or molecular tests
chromogenic substrates in this medium and show red coloured colonies
to a limited number of presumptive positive isolates (CDC, 2009).
on the plate. Non-enterococci produce colourless, blue/violet or
Finally, thanks to the continuous development of new molecules, they
turquoise colonies (Miranda et al., 2005).
also participate to highlight the microbial complexity, as illustrated by
the number of colony variants of P. aeruginosa isolated from sputum
5.2.9. C. perfringens
samples of cystic fibrosis patients (Laine et al., 2009).
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 Disclosure statement
remain smaller. C. perfringens colonies can be detected using 4-MU-
phosphate. Acid phosphatase is a highly specific indicator for C. In the last three years, Arthur James has received financial support
perfringens, which shows a light blue fluorescence on this medium. for consultancy from bioMérieux. He also receives financial remuner-
The new chromoselect CP agar, is now available, which can be used ation from sales of identification devices and a chromogenic medium
in water microbiology. C. perfringens colonies give green coloured supplied by bioMérieux.
colonies, and other clostridia species give violet, or blue coloured In the last three years, John Perry has received financial support for
colonies (Sigma, Switzerland). research or consultancy from suppliers of chromogenic culture media
including bioMérieux, Becton Dickinson and Bio-Rad. He also receives
5.2.10. L. monocytogenes financial remuneration from sales of a chromogenic medium supplied
Several studies have been performed using chromogenic media for by International Diagnostics Group (Lab M).
the detection of L. monocytogenes and are summarised by Reissbrodt David Pincus and Sylvain Orenga have been employed by bioMérieux
(2004). There are two groups of such media: the first, including Agar for the last three years. Sylvain Orenga also received financial re-
Listeria according to Ottaviani and Agosti (ALOA), utilizes cleavage by muneration for patent applications and registrations.
phosphatidylinositol-specific phospholipase C (PI-PLC) of L-α-phospha- Mammad Manafi has no potential conflict to declare.
tidylinositol, forming a white precipitation zone around the colony,
combined with the chromogenic substrate 5-bromo-4-chloro-3-indoxyl- Acknowledgment
β-D-glucopyranoside for detection of β-D-glucosidase, which occurs in
all Listeria spp. The typical colony morphology of Listeria spp. is reported The authors thank Daniel Monget for his broad and rigorous
to be turquoise blue. Pathogenic Listeria colonies are additionally involvement in the field of enzymatic substrates for microbiology
surrounded by a translucent halo. The second group of media utilizes especially for the improvement of microbial identification. By the
5-bromo-4-chloro-3-indoxyl-myoinositol-1-phosphate, forming blue- results of his work as well as by direct training of some of us, he
turquoise colonies of pathogenic Listeria spp.: L. monocytogenes and L. contributed deeply to our knowledge. And even more, he allowed us
ivanovii. A recent paper (Stessl et al., 2009) evaluated six chromogenic to meet and work together in a creative and friendly relationship. As
media similar to ALOA, in addition testing the ability to facilitate growth such, he indirectly inspired and contributed to this review.
of stressed L. monocytogenes strains and mixed cultures with competitive
non-Listeria and enumeration of L. monocytogenes in food samples. They
References
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