Enzymatic substrates in microbiology

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 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 b

a r t i c l e

i n f o

Article history: Received 19 May 2009 Received in revised form 24 July 2009 Accepted 3 August 2009 Available online 11 August 2009 Keywords: Chromogenic medium Detection Identification Synthetic enzymatic substrate

a b s t r a c t Enzymatic substrates are powerful tools in biochemistry. They are widely used in microbiology to study metabolic pathways, to monitor metabolism and to detect, enumerate and identify microorganisms. Synthetic enzymatic substrates have been customized for various microbial assays, to detect an expanding range of both new enzymatic activities and target microorganisms. Recent developments in synthetic enzymatic substrates with new spectral, chemical and biochemical properties allow improved detection, enumeration and identification of food-borne microorganisms, clinical pathogens and multi-resistant bacteria in various sample types. In the past 20 years, the range of synthetic enzymatic substrates used in microbiology has been markedly extended supporting the development of new multi-test systems (e.g., Microscan, Vitek 2, Phoenix) and chromogenic culture media. The use of such 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. 2.

3.

4. 5.

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

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☆ 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:[email protected]. ⁎ Corresponding author. Tel.: +33 4 74 95 25 43; fax: +33 4 74 95 26 43. E-mail address: [email protected] (S. Orenga). 0167-7012/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2009.08.001

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

1. Introduction 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 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 peptidases (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

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lower toxicity at pH 7.0 (Chilvers et al., 2001). Consequently, after 6 h incubation, the relative fluorescence produced by coliform bacteria growing 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 phosphate 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 surrounded 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 fluorophores or quenchers (Vencill et al., 1985).

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).

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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-3acetic 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-2deoxy-β-D-glucopyranosyloxy)-3-methoxyphenyl]-vinyl)-1-(propan3-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-4chloro-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 producing 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′-dihydroxyflavone (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-aminophenoxazin-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 Lalanyl-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-galactoside 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).

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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 phosphatidylinositolphospholipase 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 4dimethylaminobenzaldehyde (DMABA), 4-dimethylaminocinnamaldehyde (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 enterococci, 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 reagent) 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 differentiation between Y. enterocolitica, which does not hydrolyse these substrates, 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-nonulopyranosidonic 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

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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.

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a Pócsi et al., 1990; b Armstrong et al., 2001; c Pearson et al., 1963; d Rothe et al., 1992.

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(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-carboxylate than with the other 7-nitrocoumarins. 3.3. Luciferases

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-3indoxyl-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-methylcoumarin (Manafi and Kneifel, 1990) and, more recently, 9-(4′-N-[L-Ala-LAla-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 differentiate Listeria monocytogenes (negative) from the other Listeria species (positive) (Clark and McLaughlin, 1997). Detection of carboxypeptidase can be achieved by the use of Nbenzoyl-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 positive) 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 surfacedisplayed 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 chromogenic 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

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 synthetic 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-βD-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 chromophores 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), Streptococcaceae (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.

Fig. 2. Hydrolysis of synthetic enzymatic substrates.

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Table 2 Summary of targeted enzymatic activities, dyes and synthetic enzymatic substrates according to microbiological applications. Enzyme Enumeration and isolation plate medium

Glycosidase

α-Galactosidase β-Galactosidase

α-Glucosidase

β-Glucosidase

Cellobiosidase β-Glucuronidase

Esterase

Most probable number

Arylamidase/peptidase Miscellaneous Esterase Arylamidase/peptidase Miscellaneous Glycosidase

Cytometry

Glycosidase

Spot test

Multi-test identification device (detailed enzymatic activities: see Table 3)

β-Ribofuranosidase Hexosaminidase Lipase PI-PLC PC-PLC Phosphatase

β-Alanyl arylamidase Deoxyribonuclease Lipase L-Alanyl arylamidase β-Lactamase β-Glucuronidase β-Galactosidase β-Glucuronidase β-Galactosidase

Dye/substrate

Microorganism

Sample

Indoxyl Indoxyl Indoxyl Indoxyl Indoxyl Indoxyl Alizarin Indoxyl Indoxyl Indoxyl Indoxyl Indoxyl Indoxyl Indoxyl Indoxyl Indoxyl Indoxyl Indoxyl

Salmonella E. coli O157:H7 Escherichia coli E. coli O157:H7 Coliform, Staphylococcus saprophyticus VREb Cronobacter sakazakii Staphylococcus aureus MRSA VRE KESC ESBL producing enterobacteria Vibrio Enterococci VRE Listeria spp Candida kefyr, Candida lusitaniae, Candida tropicalis Cronobacter sakazakii E. coli ESBL producing E. coli Streptococcus agalactiae Yersinia enterocolitica Candida albicans, Candida dubliniensis Salmonella Listeria monocytogenes Bacillus S. aureus S. agalactiae Clostridium perfringens Candida spp. Pseudomonas aeruginosa S. aureus Salmonella Gram+/Gram− β-lactamase producing bacteria E. coli Coliform E. coli Coliform

C–Fa C–F C C–F F–W C C F C–F C C C C C–F C–W C F C

Indoxyl 4-MU, Indoxyl Indoxyl Indoxyl Indoxyl, 3′,4′-Dihydroxyflavone Indoxyl, VLPA Indoxyl Indoxyl Indoxyl Indoxyl Indoxyl 4-MU, Indoxyl Indoxyl 7-Amido-1-pentyl-phenoxazin-3-one Indoxyl 4-MU AMC, p-Nitroanilide Nitrocefin, PADAC, CENTA, Cefesone 4-MU Nitrophenol, 4-MU Fluorescein Fluorescein

F C–F–W C C C C C–F F F C–F C F C C F C–F F C F F W W

Enzyme

Dye

Microorganism

Glycosidase

Nitrophenol Naphthol Indoxyl 4-MU Resorufin

Esterase

Nitrophenol Naphthol Indoxyl 4-MU

Arylamidase/peptidase

p-Nitroanilide Naphthylamine AMC DCAP

Aerobic Gram-negative bacteria Aerobic Gram-positive bacteria Coryneforms Yeasts Anaerobes Fastidious Gram-negative Aerobic Gram-negative bacteria Aerobic Gram-positive bacteria Coryneforms Yeasts Anaerobes Fastidious Gram-negative Aerobic Gram-negative bacteria Aerobic Gram-positive bacteria Coryneforms Yeasts Anaerobes Fastidious Gram-negative

a

C, clinical; F, food; W, water. VRE, vancomycin-resistant enterococci; MRSA, methicillin-resistant Staphylococcus aureus; ESBL, extended spectrum ß-lactamase; KESC, Klebsiella, Enterobacter, Serratia, Citrobacter. b

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.

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S. Orenga et al. / Journal of Microbiological Methods 79 (2009) 139–155 Table 3 (continued)

Table 3 Enzymatic tests and applications in commercial ID products. Enzyme

Glycosidases N-acetyl-β-galactosaminidase N-acetyl-β-glucosaminidase α-Amylase α-Arabinofuranosidase α-Arabinosidase β-Cellobiosidase β-Chitobiosidase α-Fucosidase β-Fucosidase α-Galactosidase β-Galactosidase α-Glucosidase β-Glucosidase or esculin hydrolysis β-Glucuronidase α-Maltosidase β-Maltosidase α-Mannosidase β-Mannosidase β-Xylosidase Arylamidases Alanyl arylamidase β-Alanyl arylamidase Alanyl-alanine arylamidase Alanyl-phenylalanyl-proline arylamidase Arginine arylamidase Arginyl-arginine arylamidase Aspartic acid arylamidase Citrulline arylamidase α-Glutamic acid arylamidase γ-Glutamyl transferase Glutamyl-glutamic arylamidase Glutamyl-glycyl-arginine arylamidase Glycine arylamidase Glycyl-arginine arylamidase Glycyl-glycine arylamidase Glycyl-proline arylamidase Glycyl-tryptophan arylamidase Histidine arylamidase Hydroxyproline arylamidase Isoleucine arylamidase Leucine arylamidase Leucyl-glycine arylamidase Lysine arylamidase Lysyl-alanine arylamidase Methionine arylamidase Phenylalanine arylamidase Proline arylamidase Pyrrolidonyl arylamidase Serine arylamidase Seryl-tyrosine arylamidase Tryptophan arylamidase Tyrosine arylamidase Esterases Acetate esterase Lipase Phosphatase Phospholipase Miscellaneous Arginine dihydrolase Gelatinase Hippurase Glutamic acid decarboxylase Indole formation (tryptophanase) Lysine decarboxylase Nitrate reductase Ornithine decarboxylase Phenylalanine deaminase

Enzyme

Application GNa

GP

YS

AC

Xb X

X X

X X X

X

X

X X X

FN

X X X X

X X

X

X

X

X X X X X X X

X X

X X X X

X

X X

X

X

X X

X

X

X X

X

X

X

X X X X

X X X

X

X

X X

X X

X

X

X X X X

X

X X X X

X

X

X X

X X

X X

X X X X

X X

X

X X X X X X X X

X

X X

X X

X

X X

X

X

X X X

X

X X

X

X

X X X

X X

X X X X X X

X X X

X X

YS

AC

FN

X X

X

X

X X

X

GN, non-fastidious Gram-negative; GP, aerobic Gram-positive except coryneforms; YS, yeasts; AC, anaerobes/coryneforms; FN, fastidious Gram-negative. b X, used in corresponding identification reagent.

X

The differential capabilities afforded by enzymatic tests have enabled development of highly accurate ID methods surpassing those available previously with only carbon and nitrogen substrate utilization tests. Additionally, an increased use of polyphasic, i.e., phenotypic, genotypic, and chemotaxonomic, characterization (Vandamme et al., 1996) has resulted in better-characterized culture collections and stronger knowledge bases also reinforcing the development of highly accurate phenotypic ID products. Currently, there are many commercial ID products with essentially equivalent performances and other features, e.g., workflow, cost, etc., are key for their selection in various laboratories. Commercial ID product evaluations typically show a wide range of accuracy most often due to differences in study design. Influential factors of the study design include species and strain selection, comparative method(s), database version, etc. It is therefore prudent to focus on several studies rather than a single one. While it is impractical to review all available products, the tables below focus on some of the more widely used products with enzymatic substrates and their respective performances. It is noteworthy that some recent evaluations (e.g., Heikens et al., 2005, Layer et al., 2006, Sanguinetti et al., 2007, Delmas et al., 2008) compare ID products to molecular methods while older evaluations compare ID products to conventional methods (e.g., Ruoff and Kunz, 1983, Facklam et al., 1985, O'Hara and Miller, 1992) or to other commercial products (e.g., Endimiani et al., 2002, Stefaniuk et al., 2003, Donay et al., 2004). One cautionary note is that comparison of one commercial product to another commercial product may compound an inherent error rate present in the comparator method. These and other factors can account for wide differences in observed performances as shown in Tables 4–8. 5. Application of enzyme substrates in culture media for microbiology

X

X X

GP

X

X X X X

X

GNa

a

X X

X X X X X

X X

Miscellaneous Pyrazinamidase Tryptophan deaminase Urease

Application

X X X

Over the last 20 years, there has been a rapid expansion in the development and commercial availability of chromogenic agar media for the detection of pathogenic bacteria and yeasts (Perry and Freydière, 2007). Such culture media typically contain multiple substrates that allow bacteria to form coloured colonies based on their enzymatic activity. This facilitates the differentiation of species within polymicrobial cultures and the targeting of pathogens with high specificity. When specific pathogens are targeted, selective agents such as antibiotics are employed to limit the number of species able to grow. Most media rely upon the inclusion of indoxylic substrates in order to generate colonies with contrasting colours. For example, the release of green and red chromogens from two distinct substrates can result in the formation of green, red or purple colonies depending on whether one or both enzyme activities are present. Pathogens may therefore be differentiated from commensal bacteria by their possession of either one or both enzymes. Indoxylic glycosidase substrates are employed in the majority of chromogenic media used in microbiology laboratories including media for the detection of urinary tract pathogens, Salmonella spp., E. coli O157:H7, Cronobacter sakazakii, Vibrio, S. aureus, enterococci, group B streptococci, Listeria and yeasts. The glycosidases targeted are largely confined to a narrow range, which includes α- or β-galactosidase, α-

S. Orenga et al. / Journal of Microbiological Methods 79 (2009) 139–155 Table 4 Products for Enterobacteriaceae (E) and/or non-Enterobacteriaceae (NE). No. isolates

E NE E/NE E E/NE E/NE E/NE E/NE NE NE

99.2 99.0 98.6 89.1 94.0 95.5 88.7 87.9 91.6 97.0

229 123 201 545 524 379 125 345 414

NE NE NE E/NE E/NE E/NE E NE E

94.8 95.1 74.6 92.8 77.3 96.8 92.0 96.2 95.4

Hayek and Willis, 1976 Appelbaum et al., 1984 Robinson et al., 1995 Wauters et al., 1995 Holmes et al., 1994 Robinson et al., 1995 Wauters et al., 1995 O'Hara et al., 1997 Appelbaum and Leathers, 1984 von Graevenitz and Zollinger-Iten, 1985 Martin et al., 1986 Palmieri et al., 1988 Wauters et al., 1995 Monnet et al., 1994 O'Hara and Miller, 1999 Kitch et al., 1994 Lee et al., 1994 Kitch et al., 1992 Freney et al., 1991b

Automated products MicroScan Rapid 383 Neg Combo 3 467 252 MicroScan Rapid 652 Neg ID3 511 134 Phoenix NID 136 174 187 564 251 VITEK GNI+ 619 304 299 454 VITEK 2 GN 655 331 426 416

E/NE E/NE E/NE E/NE E/NE NE E/NE E/NE E/NE E/NE E E/NE E/NE NE E E/NE E/NE E/NE E/NE

95.3 91.9 96.4 94.0 88.8 93.3 95.6 93.7 93.0 89.4 94.4 87.6 94.4 92.3 88.8 99.4 97.0 99.5 97.1

Pfaller et al., 1991 O'Hara and Miller, 1992 O'Hara et al., 1993 Bascomb et al., 1997 O'Hara and Miller, 2000 O'Hara and Miller, 2002 Endimiani et al., 2002 Stefaniuk et al., 2003 Donay et al., 2004 O'Hara, 2006 Carroll et al., 2006b O'Hara et al., 1997 Bourbeau and Heiter, 1998 Sung et al., 2000 O'Hara and Miller, 2003 Funke and Funke-Kissling, 2004 Wallet et al., 2005 Renaud et al., 2005 Schreckenberger et al., 2005

ID 32 E RapID onE RapID NF Plus rapid ID 32E

Table 5 Products for Micrococcaceae and/or Streptococcaceae.

Type of % Reference taxa tested Correct

Manual products api 20E 245 258 512 505 Crystal E/NF 266 512 706 626 api 20 NE 262 431

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No. Type of taxa isolates tested Manual products api Staph 120 212 200 58 api 20 Strep 209 77 251 119 199 Crystal GP 191 124 120 ID 32 Staph 440 200 137 rapid ID 32 433 Strep 156 122 124 RapID STR 266 247

CNSa Staphylococci CNS CNS Streptococci Viridans Streptococci Viridans Streptococci All GPC Enterococci CNS CNS CNS CNS Streptococci and related Viridans Streptococci/enterococci Enterococci Streptococci Streptococci

Automated products MicroScan 283 CNS Pos ID2 202 Enterococci 104 Staphylococci MicroScan 239 Staphylococci Rapid Pos 233 CNS 285 CNS Phoenix PID 118 All GPC 58 CNS 410 Staphylococci/enterococci 110 CNS 200 Streptococci/enterococci VITEK GPI 507 Streptococci 495 CNS 374 Enterococci VITEK 2 GP 364 All GPC 249 All GPC 121 Enterococci 110 CNS 168 Staphylococci 120 CNS a

% Reference Correct 88.3 86.8 85.5 87.9 85.2 90.0 86.1 92.0 88.4 90.0 82.0 67.5 95.2 79.5 91.2 95.3 87.2 93.0 85.0 97.0 93.9

Giger et al., 1984 Baldellon and Mégraud, 1985 De Paulis et al., 2003 Heikens et al., 2005 Appelbaum et al., 1984 Etienne et al., 1984 Facklam et al., 1984 Ruoff and Kunz, 1983 Watts, 1989 von Baum et al., 1998 Jackson et al., 2004 Kim et al., 2008 Ieven et al., 1995 Renneberg et al., 1995 Edwards et al., 2001 Freney et al., 1992 Kikuchi et al., 1995 Gorm Jensen et al., 1999 Jackson et al., 2004 Appelbaum et al., 1986 You and Facklam, 1986

97.5 81.2 94.2 95.4 76.0 88.7 89.8 62.1 99.3 80.0 92.0 87.8 85.9 91.4 94.5 94.4 100.0 90.9 87.5 95.0

Weinstein et al., 1998 Iwen et al., 1999 Sáa et al., 1999 Stoakes et al., 1992 Grant et al., 1994 Weinstein et al., 1998 Donay et al., 2004 Heikens et al., 2005 Carroll et al., 2006a Layer et al., 2006 Brigante et al., 2006 Facklam et al., 1985 Bannerman et al., 1993 Willey et al., 1993 Funke and Funke-Kissling, 2005 Wallet et al., 2005 Abele-Horn et al., 2006 Layer et al., 2006 Delmas et al., 2008 Kim et al., 2008

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 substrates for the detection of phosphatase or esterase activity in order to target specific pathogens.

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 differentiate E. coli and highlight β-glucosidase producers such as Klebsiella, Enterobacter, 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

substitute the β-glucuronidase substrate for a β-galactosidase substrate. 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-Dgalactosidase-positive coliforms, Salmonella strains give red colonies because of their ability to produce acid from propylene glycol revealed

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Table 6 Products for yeasts.

Table 8 Products for Neisseria (N), HACEK (H) group, and Campylobacter (C). No. isolates

Manual products api Candida

RapID Yeast Plus

Automated products MicroScan Rapid Yeast VITEK YBC

VITEK 2 YST

% Correct

Reference

619 198 159 202 300 447 133 201 225 750

95.8 91.4 91.8 97.0 99.3 97.6 94.0 96.0 87.1 95.5

Fricker-Hidalgo et al., 1996 Bernal et al., 1998 Campbell et al., 1999 Paugam et al., 1999 Kitch et al., 1996 Espinel-Ingroff et al., 1998 Heelan et al., 1998 Wadlin et al., 1999 Smith et al., 1999 Sanguinetti et al., 2007

401 357 150 1106 221 150 398 409 97 623 750 136

91.8 96.6 67.0 94.3 83.3 85.0 97.2 89.7 99.0 98.6 98.4 94.1

Land et al., 1991 St.-Germain and Beauchesne, 1991 Riddle et al., 1994 Land et al., 1984 Pfaller et al., 1988 Riddle et al., 1994 el-Zaatari et al., 1990 Fenn et al., 1994 Aubertine et al., 2006 Hata et al., 2007 Sanguinetti et al., 2007 Valenza et al., 2008

by neutral red as the pH indicator. SM ID also incorporates a β-Dgalactosidase substrate and glucuronic acid, which is metabolised by Salmonella spp. In most of the second generation of chromogenic media, Salmonella is typically targeted by utilizing a substrate for C8 esterase activity. Few other species of Enterobacteriaceae produce C8 esterase and these may be differentiated by incorporation of a substrate for β-galactosidase, or preferably β-glucosidase so that the detection of lactose fermenting Salmonella is not precluded. Indoxylic esters (e.g., octanoate or nonanoate) are normally used as substrates but other chromogens have been utilized (Cooke et al., 1999; Gaillot

Table 7 Products for anaerobes and coryneforms.

Manual products Crystal ANR ID 32 A

RapID ANA II api Coryne

Crystal GP RapID CB Plus

VITEK ANI Automated products MicroScan Rapid Anaerobe VITEK 2 ANC

No. isolates

% Correct

Reference

322 214 105 102 460 122 300 566 240 177 160 407 178 50 50 86 378 50 341

81.7 96.3 86.3 96.1 82.0 95.1 87.0 78.0 97.6 88.1 87.5 90.5 91.0 72.0 76.0 80.2 80.9 52.0 83.3

Cavallaro et al., 1997 Kitch and Appelbaum, 1989 Grollier et al., 1992 Pattyn et al., 1993 Downes et al., 1999 Sperner et al., 1999 Celig and Schreckenberger, 1991 Marler et al., 1991 Freney et al., 1991a Gavin et al., 1992 Soto et al., 1994 Funke et al., 1997 Almuzara et al., 2006 Adderson et al., 2008 Adderson et al., 2008 Hudspeth et al., 1998 Funke et al., 1998 Adderson et al., 2008 Schreckenberger et al., 1988

237

76.7

Stoakes et al., 1990

365 251

95.1 92.0

Rennie et al., 2008a Mory et al., 2009

No. Type of taxa % Correct Reference isolates tested Manual products RapID NH 240 187 299 90 380 api Campy 100 62 87 api NH 305 380 VITEK NHI 480 380

N H H biotyping N H biotyping C C C N/H H biotyping N/H H biotyping

97.9 89.8 92.0 94.4 51.6 92.0 79.0 94.3 99.7 97.6 95.2 95.0

Robinson and Oberhofer, 1983 Doern and Chapin, 1984 Doern and Chapin, 1987 Philip and Garton, 1985 Munson and Doern, 2007 Huysmans et al., 1995 Reina et al., 1995 Shih, 2000 Barbé et al., 1994 Munson and Doern, 2007 Janda et al., 1987 Munson and Doern, 2007

Automated products MicroScan HN 423 VITEK 2 NH 188 371

N/H N/H/C N/H/C

94.6 96.8 96.5

Janda et al., 1989 Valenza et al., 2007 Rennie et al., 2008b

et al., 1999). An alternative means of detecting Salmonella is exploited in ABC medium, which utilizes an indoxylic α-galactoside (hydrolysed by Salmonella) in combination with 3,4-cyclohexenoesculetin-β-Dgalactoside (Perry et al., 1999). In order to improve the selectivity of media for Salmonella, ‘suicide substrates’ such as alafosfalin have been exploited (Perry et al., 2002). Such substrates release toxic molecules when hydrolysed so that commensal bacteria may be selectively inhibited on the basis of their ability to accumulate and hydrolyse them. E. coli O157:H7 may also be recovered from stool samples using specific chromogenic media designed for food samples (see below). 5.1.3. S. aureus S. aureus is the most common cause of wound infection and diagnosis 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 staphylococci necessitating the use of complementary glycosidase substrates, which are not hydrolysed by S. aureus, e.g., an indoxylic βglucoside. The use of a phosphatase substrate is exploited in several commercially available chromogenic media including CHROMagar Staph aureus (Gaillot et al., 2000). An alternative approach is to target α-glucosidase activity as employed in S. aureus ID (Perry et al., 2003). 5.1.4. Differentiation of yeasts When different species of yeast are present in a polymicrobial culture, they are extremely difficult to distinguish from each other using conventional media. Chromogenic media have been designed to provide differentiation of species and identification of the most important pathogenic yeast, Candida albicans. The latter is achieved by the incorporation of a β-hexosaminidase substrate, which is hydrolysed by C. albicans to generate coloured colonies. CHROMagar Candida employs a further substrate for phosphatase activity allowing the specific identification of Candida tropicalis, which produces both enzymes (Odds and Bernaerts, 1994). 5.1.5. Group B Streptococcus During antenatal screening many women are routinely screened for the presence of Streptococcus agalactiae (group B Streptococcus) as a vaginal commensal since this species may cause severe disease in neonates. S. agalactiae may be differentiated from other vaginal flora by use of an indoxylic phosphate, which is hydrolysed to produce 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 infectioncontrol 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 authorities 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 αglucosidase and β-galactosidase to allow detection and differentiation of the two most important species; Enterococcus faecalis and Enterococcus 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 aminopeptidase 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 substrates 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 fluorescence 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 rhamnose 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 Chromogenic 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, Salmonella 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-indolylα-D-glucopyranoside (X-α-Glc) is added to a basal medium to differentiate C. sakazakii strains from other members of the Enterobacteriaceae. 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 supplemented 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

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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), BioChrome 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 detection 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 inhibits the accompanying bacterial flora and causes the colonies to remain smaller. C. perfringens colonies can be detected using 4-MUphosphate. 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-phosphatidylinositol, forming a white precipitation zone around the colony, combined with the chromogenic substrate 5-bromo-4-chloro-3-indoxylβ-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 blueturquoise 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 simultaneously descriptive and functional information. For the detection of carbapenemase producing Enterobacteriaceae, they allow for a reduction 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 remuneration 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 remuneration 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. References Abele-Horn, M., Hommers, L., Trabold, R., Frosch, M., 2006. Validation of VITEK 2 version 4.01 software for detection, identification, and classification of glycopeptideresistant enterococci. J. Clin. Microbiol. 44, 71–76. Adderson, E.E., Boudreaux, J.W., Cummings, J.R., Pounds, S., Wilson, D.A., Procop, G.W., Hayden, R.T., 2008. Identification of clinical coryneform bacterial isolates:

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