24/08/2019
The application of chromogenic media in clinical microbiology
J.D. Perry A.M. Freydière
Introduction
The traditional approach to the detection of pathogenic bacteria in pathological specimens has typically involved the inoculation of one or more general purpose culture media such as Blood agar. Such media allow the growth of a wide range of bacteria, and suspect pathogens are detected on the basis of their colonial appearance (e.g. pigmentation, morphology, haemolysis). Such colonial characteristics rarely permit more than a presumptive identification and biochemical and/or serological tests are required for definitive identification. This approach frequently necessitates the testing of commensal bacteria that may resemble pathogens. For example, one of the commonest pathogens sought from infections of skin and wounds is Staphylococcus aureus and many commensal staphylococci of the skin may need to be tested to exclude the presence of this pathogen. Detection of Salmonella spp., a frequent cause of gastroenteritis, requires isolation of the pathogen from stool samples. Media containing lactose, plus a pH indicator, have been traditionally used for differentiation of Salmonella (a nonfermentor) from commensals such as Escherichia coli. However, it is frequently necessary to screen many other commensals that also fail to ferment lactose (e.g. Proteus spp.) to exclude the presence of Salmonella. The screening of commensal bacteria to exclude pathogens is time consuming and can be costly in terms of serological or biochemical reagents.
Over the last 20 years, a range of chromogenic media has been developed that are designed to target pathogens with high specificity. Such media exploit enzyme substrates that release coloured dyes upon hydrolysis, thus resulting in pathogens forming coloured colonies that can easily be differentiated from commensal flora. Ideally, commensal bacteria should either be inhibited completely by selective agents or form colourless colonies thus allowing pathogens to ‘stand out’ against background flora. It is rare, however, for a pathogen to exclusively produce any particular enzyme and it is customary for a second complementary chromogenic substrate to be incorporated causing some commensal bacteria to generate either a second colour or a combination of both colours, thereby providing differentiation from the target pathogen.
Chromogenic media are principally designed to limit the requirement for screening commensal organisms to exclude the presence of pathogens, thus saving time and reagents. Chromogenic media may also afford an increased sensitivity, as coloured colonies can be detected more easily when present amongst other flora. This is often augmented by improvements in selectivity, when compared with conventional culture media, thus limiting the amount of commensal flora able to grow. Here, we review the various applications of chromogenic media and their performance when compared with traditional culture media for the isolation and detection of various pathogenic bacteria and yeasts.
Chromogenic substrates
A wide range of coloured molecules or ‘chromogens’ has been derivatized to produce enzyme substrates that release the coloured product following hydrolysis by a specific enzyme. For the purposes of this review, discussion will be restricted to those that have been applied in agar media for the detection of pathogenic bacteria and fungi. An ideal chromogenic substrate should be hydrolysed to release a coloured product that remains highly localized on microbial colonies. This allows clear differentiation of microbes producing the target enzyme from those that do not. This is especially important when attempting to detect specific pathogens within polymicrobial cultures. The substrate and products of hydrolysis should be noninhibitory to microbial growth.
Most commercially available chromogenic media have exploited indoxylic substrates. Indoxyl, and its halogenated derivatives, can be derivatized to form a range of esters. Release of indoxyl via hydrolysis by a specific bacterial enzyme results in the formation of brightly coloured indigo dye. This is due to spontaneous dimerization of indoxyl molecules in the presence of oxygen. Halogenation of the indoxyl molecule has a dramatic effect on the colour and intensity of this chromogen. For example, 5‐bromo‐4‐chloro‐indoxyl forms a bright green/blue dye whereas 5‐bromo‐6‐chloro‐indoxyl forms a magenta dye. Indoxylic glycosides including glucoside, galactoside and glucuronide derivatives are widely used because of their high sensitivity, low toxicity and availability from a number of commercial sources.
Other core molecules have been exploited in the design of chromogenic substrates. One particularly useful group of molecules comprises metal chelators. Esculin is a naturally occurring glycoside and a useful substrate for detection of β‐glucosidase. Hydrolysis of esculin leads to release of 6,7‐dihydroxycoumarin (esculetin), which binds iron to form a brown/black complex. The application of esculin in agar media is limited by diffusion of the coloured complex. A modified esculetin derivative, 3,4‐cyclohexenoesculetin, was synthesized and derivatized to form glycosides (James et al. 1996, 1997) that have been utilized in agar media (Perry et al. 1999; Smith et al. 2001). Hydrolysis of these substrates releases 3,4‐cyclohexenoesculetin, which, in the presence of iron, forms a black chelate, which remains highly localized within bacterial colonies and does not diffuse through agar.
8‐Hydroxyquinoline is another metal chelator, which has been derivatized to form glycosides (James and Yeoman 1987, 1988; James et al. 1996) that have been exploited in agar media (Dalet and Segovia 1995; Larinkari and Rautio 1995). When released by hydrolysis, 8‐hydroxyquinoline binds with iron to form a brown/black chelate. Toxicity of this chelate towards Gram‐positive bacteria has precluded the more widespread application of such substrates. Other chelation‐based substrates applicable to agar media include those based on alizarin and 3′, 4′‐dihydroxyflavone although these have not yet been applied in commercially available chromogenic media (James et al. 2000; Butterworth et al. 2004).
The synthesis of a series of extended phenolics has been reported by Aamlid et al. (1990) and closely related compounds have since formed the basis of chromogenic substrates for the detection of glycosidases and esterases. These have been applied for the detection of Candida spp. (Cooke et al. 2002) and Salmonella spp. (Cooke et al. 1999). The attributes of a range of chromogens derivatized as substrates for β‐glucosidase have recently been compared and their chemical structures illustrated (Perry et al. 2007).
Detection of urinary tract pathogens
Kilian and Bülow (1979) described a chromogenic medium that employed a substrate for β‐glucuronidase, thus allowing the direct detection of the commonest pathogen of the urinary tract, Escherichia coli. In a large study using 9247 urine samples, the authors reported that 94% of E. coli strains could be identified by their coloured appearance alone and no E. coli strains were misidentified. When compared with the use of conventional media (Blood and MacConkey agars), the authors reported a cost reduction of 46% for media, and a 67% reduction in the time required for processing samples. In this study, as in most others, E. coli accounted for the majority (58%) of isolates with other Enterobacteriaceae (24%) and enterococci (11%) also isolated frequently. The cost‐effectiveness of using chromogenic media for urine culture because of decreased labour time and lower usage of reagents has been demonstrated by others (D’Souza et al. 2004).
As illustrated above, amongst the Enterobacteriaceae, E. coli are the only β‐glucuronidase producers that are likely to be found in urine samples. Mazoyer et al. (1995) reported the evaluation of CPS ID2 (CPS: Coli‐Proteus‐Strep Identification). In addition to a β‐glucuronidase substrate, this medium employs a substrate for detection of β‐glucosidase activity in enterococci and the Klebsiella/Enterobacter/Serratia group, as well as tryptophan for demonstration of tryptophan deaminase in the Proteus‐Providencia‐Morganella (PPM) group. The medium allowed direct identification of the commonest urinary tract pathogens on the basis of colony colour without the need for laborious confirmatory tests. Over recent years a range of media, based on similar principles, has been made commercially available. These media generally employ either β‐glucuronidase or β‐galactosidase for the identification of E. coli, tryptophan for the identification of the PPM group and a β‐glucosidase substrate for the detection of enterococci. The use of a β‐galactosidase substrate reduces the specificity of identification for E. coli and some species, e.g. Citrobacter freundii may be misidentified as E. coli unless supplementary biochemical tests (e.g. for indole production) are employed (Aspevall et al. 2002; Fallon et al. 2002; Perry et al. 2003a).
Several authors have compared the performance of these chromogenic media against each other and against conventional agars. Carricajo et al. (1999) reported a comparative evaluation of five chromogenic media using 443 urine samples and found that CPS ID2 and CHROMagar Orientation demonstrated the best performance. Aspevall et al. (2002) compared four chromogenic media along with cystine lactose electrolyte deficient (CLED) agar and MacConkey agars with 1200 urine samples. They concluded that the chromogenic media were slightly better than the conventional media because of their superior ability to differentiate mixed cultures. This point has been supported by other studies, e.g. Fallon et al. (2003) compared three chromogenic agars with traditional CLED agar and found that all three detected significantly more mixed growths than CLED agar (P < 0·01). Similary, Perry et al. (2003a) showed that, of 168 confirmed mixed cultures, only 78% were recognized as polymicrobial on CLED medium compared with 99% on Uriselect 4 and 97% on CPS ID2.
A wide range of complementary tests has been recommended for use with chromogenic media including tests for indole production, pyrrolidonyl peptidase, urease and lysine or ornithine decarboxylases. Such tests are used either to increase the specificity of E. coli identification, or to broaden the range of species that may be identified with the assistance of chromogenic media (Carricajo et al. 1999; Ohkusu 2000;Fallon et al. 2002).
Detection of Staphylococcus aureus
Isolation of Staph. aureus is usually achieved by culture of specimens on general purpose media, such as blood agar, and subsequent identification of suspect colonies using biochemical and/or serological tests. Most commonly this involves testing colonies of staphylococci for agglutination with sensitized latex particles to detect bound coagulase, protein A and/or specific capsular antigens (van Griethuysen et al. 2001).
Chromogenic media have been designed for the isolation and detection of Staph. aureus with some success (Gaillot et al. 2000; Perry et al. 2003b). CHROMagar Staph aureus is a selective agar medium that employs a combination of chromogenic enzyme substrates. Staphylococcus aureus strains grow as mauve colonies on this medium whereas most other staphylococci produce white, or occasionally, blue colonies. Gaillot et al. (2000) examined the performance of CHROMagar Staph aureus with 2000 clinical samples and reported a sensitivity of 95·5%. This was significantly higher than the sensitivity of blood agar (81·9%) after 24 h incubation (P < 0·001). The specificity of the medium was also high as 97·4% of mauve colonies were confirmed as Staph. aureus. In another study with 775 clinical samples, CHROMagar Staph aureus showed a sensitivity of 98·5% compared with 91·8% using blood and chocolate agar plates (Carricajo et al. 2001). In this study, 91·3% of mauve colonies were confirmed as Staph. aureus and specificity could be increased to 100% using the StaphyCHROM coagulase test on mauve colonies.
S. aureus ID. is another recently described chromogenic medium for the specific detection of Staph. aureus. On S. aureus ID. colonies of Staph. aureus form distinctive green colonies because of production of α‐glucosidase. Other staphylococci form white or, occasionally, pink colonies due to the hydrolysis of a substrate for β‐glucosidase. In a study with 350 wound swabs, 96·8% of Staph. aureus strains were isolated on S. aureus ID. as green colonies compared with an isolation rate of 91·1% for CHROMagar Staph aureus and 94·3% for aztreonam blood agar after 18–20 h incubation (Perry et al. 2003b). S. aureus ID was found to be particularly effective for the inhibition of enterococci and an analysis of the data indicated that this may have proved an advantage over the other test media. Both chromogenic media were highly specific. A number of complementary tests may be used with chromogenic media, for example coagulase detection to confirm identification (Carricajo et al. 2001) or detection of methicillin resistance using latex agglutination (Merlino et al. 2000).
Detection of methicillin resistant Staphylococcus aureus
Methicillin‐resistant Staph. aureus (MRSA) is a nosocomial pathogen of world‐wide importance and an increasingly frequent cause of community‐acquired infection. A wide range of culture methods has evolved for the detection of MRSA (Davies et al. 2000; Safdar et al. 2003). Such methods have traditionally employed selective media supplemented with oxacillin or methicillin to suppress the growth of methicillin‐sensitive Staph. aureus (MSSA). Mannitol salt agar supplemented with oxacillin is widely used (Gorss 1992) but has shown limited sensitivity and specificity in some studies (Davies and Zadik 1997; Davies et al. 2000; Simor et al. 2001; Gurran et al. 2002). A modified version of mannitol salt agar is Oxacillin Resistance Screening Agar base (ORSAB), which is more selective due to the presence of lithium chloride and polymyxin, and contains aniline blue as a pH indicator (Simor et al. 2001). Some studies have revealed similar limitations regarding sensitivity and specificity (Apfalter et al. 2002; Becker et al. 2002; Blanc et al. 2003).
Ciprofloxacin is a useful agent for the suppression of MSSA strains and has been used successfully to supplement Baird‐Parker medium (Davies et al. 2000) and mannitol broth (Gurran et al. 2002). These methods are limited; however, as they cannot detect ciprofloxacin sensitive MRSA which may occur in some areas (Jones et al. 2003).
Two studies have reported the adaptation of CHROMagar Staph aureus for the isolation of MRSA by inclusion of methicillin or oxacillin. Merlino et al. (2000) examined the inclusion of either agent and found the adapted media could support growth of multidrug resistant MRSA strains but were inhibitory for some community‐acquired MRSA strains. Kluytmans et al. (2002) examined the utility of CHROMagar Staph aureus with 4 mg l−1 oxacillin and showed that the medium had limited sensitivity for MRSA after 24 h incubation. Neither of these studies examined the utility of these media with pathological samples.
There has been renewed interest in the use of cefoxitin for differentiation of MRSA from MSSA. Flayhart et al. (2005) compared the performance of a commercially available product, BBL CHROMagar MRSA (BD Diagnostics) containing 6 mg l−1 of cefoxitin, with blood agar for the isolation of MRSA from 2015 nasal swabs. The chromogenic medium recovered 95·2% of MRSA strains compared with 86·9% recovered on blood agar (P < 0·01). Cefoxitin has also been incorporated into S. aureus ID to formulate a chromogenic medium (MRSA ID) specific for MRSA. In one study with 747 clinical samples, MRSA ID showed a sensitivity of 80% after 22–24 h incubation compared with 59% and 62% for CHROMagar MRSA and ORSAB respectively (Perry et al. 2004). The specificity of the medium was also high in comparison with CHROMagar MRSA and ORSAB. The results of this study suggested that MRSA strains grow much more readily in the presence of cefoxitin compared with oxacillin possibly due to enhanced induction of the penicillin binding protein, PBP2’, by cefoxitin. In a multicentre study, Bendridi et al. (2006) compared MRSA ID with CHROMagar MRSA with 910 clinical samples. The authors reported an equivalent performance of the media after 24 h of incubation but a slightly higher sensitivity for MRSA ID after 48 h. However, in this multicenter study, a high proportion of strains isolated in one laboratory in Vienna required 48 h of incubation to generate coloured colonies on either medium.
Other chromogenic media have recently been made available for detection of MRSA. Ben Nsira et al. (2006) evaluated the performance of MRSA Select with 666 screening samples and reported a sensitivity of 99% and a specificity of 99·8% after 24 h incubation. The performance was superior to that of ORSAB and mannitol salt agar plus oxacillin. Stoakes et al. (2006) evaluated MRSA Select with 2125 screening swabs and reported a recovery of 97·3% of strains compared with 82·9% of strains using CHROMagar MRSA. The authors also showed that media containing cefoxitin were significantly better for detection of MRSA from nasal swabs than mannitol salt agar supplemented with oxacillin (P < 0·001). Using 634 screening swabs, Banavathi et al. (2006) compared MRSA Select with Oxoid MRSA Chromogenic medium, which employs cefoxitin for inhibition of MSSA and a phosphatase substrate for detection of MRSA as blue colonies. A total of 92·1% of MRSA strains were recovered on MRSA Select compared with 88·9% on the Oxoid medium.
Detection of Salmonella
A range of chromogenic media has been developed for the detection of Salmonella spp. in stool samples. Most of these media have been designed using similar principles. Rambach agar and Salmonella Detection and Identification medium (SM‐ID) were the first media of this type. In common with several subsequently developed media, Rambach agar employs a chromogenic substrate for β‐galactosidase causing common commensals such as E. coli to generate blue colonies. The vast majority of Salmonella spp. isolated from humans do not produce β‐galactosidase and are highlighted instead as red colonies because of their ability to acidify neutral red by fermentation of propylene glycol (Rambach 1990). SM‐ID also incorporates a β‐galactosidase substrate and glucuronic acid, which is fermented by Salmonella spp. (Poupart et al. 1991).
Alpha–beta chromogenic medium (ABC medium) also utilises a β‐galactosidase substrate and Salmonella are visualized by their ability to hydrolyse a second chromogenic substrate for alpha galactosidase (Perry et al. 1999). When compared with conventional agars, the high specificity of ABC medium has been shown to offer a highly cost‐effective means of detecting Salmonella spp. (O’Neill et al. 2003). In a large study with 2409 stool samples, Nye et al. (2002) confirmed the high specificity of ABC medium but reported a low sensitivity as a primary plating medium, when compared with conventional agars, due to overgrowth of commensal flora (P < 0·01). Perry et al. (2002) evaluated ABC medium in a multicentre study on three continents and found that 45% of Salmonella isolates from Burkina Faso, Africa, failed to produce alpha galactosidase and would therefore not be routinely detected. This emphasises the need to vaidate the performance of chromogenic media in different geographical locations.
Few species of Enterobacteriaceae apart from Salmonella spp. are able to cleave fatty acid esters of 7–10 carbon atoms (e.g. octanoate derivatives). In recent years a range of media has been manufactured that rely on the detection of such activity in Salmonella spp., using chromogenic esters (Cooke et al. 1999; Gaillot et al. 1999; Cassar and Cuschieri 2003). Other species that produce such esterases, such as Pseudomonas spp. and yeasts may be inhibited by incorporation of selective agents such as cefsulodin and amphotericin, respectively (Gaillot et al. 1999; Eigner et al. 2001). Eigner et al. (2001) compared one such medium, BBL CHROMagar Salmonella, with Hetkoen enteric agar using 439 stool samples. The authors reported a higher sensitivity and specificity for the chromogenic medium and recommended it as a primary isolation medium.
Chromogenic media offer a much higher degree of specificity than conventional media which are based on absence of lactose fermentation within Salmonella and/or their ability to generate hydrogen sulphide. However, some reports have shown that conventional media offer a higher degree of sensitivity particularly when specimens are plated directly onto agar media (Dusch and Altwegg 1993, 1995; Monnery et al. 1994; Nye et al. 2002; Perez et al. 2003). This is largely explained by findings showing that some chromogenic media for Salmonella spp. exhibit very poor selectivity against common commensals such as E. coli (Perry et al. 2002). An innovative approach to the inhibition of Enterobacteriaceae other than Salmonella involves the application of enzyme substrates, which release a toxic product upon hydrolysis (Druggan 2002). This approach permits the rationale design of inhibitors selecting for bacteria that lack a particular enzyme or show a reduced uptake of the ‘suicide substrate’. Such an approach may increase the selectivity of chromogenic agar, as demonstrated by the inclusion of alafosfalin in ABC medium (Perry et al. 2002).
Following enrichment in selenite broth, Salmonella spp. predominate and their detection can be conveniently confirmed with high specificity using chromogenic media (Dusch and Altwegg 1993). Direct inoculation of solid media remains necessary for primary diagnosis in order to facilitate early detection of Salmonella and because of the susceptibility of some strains to selective enrichment procedures (Patil and Parhad 1986). A further limitation of chromogenic media is that other enteric pathogens, e.g. Shigella spp. are not detected and complementary media are therefore required for routine examination of stool samples.
Detection of Escherichia coli O157
Escherichia coli O157 : H7 is the predominant clone associated with outbreaks of haemorrhagic colitis and haemolytic uraemic syndrome. Although outbreaks are uncommon, symptoms may be severe, leading to death, and in the UK this has led the Subcommittee of the PHLS Advisory Committee on Gastrointestinal Infections (2000) to recommend culture of all diarrhoeal stools for E. coli O157. Several chromogenic media have been applied to the detection of E. coli O157 from stool samples. Most are based on similar principles; relying on nonfermentation of sorbitol and/or rhamnose and lack of β‐glucuronidase activity in the commonest serotype, E. coli O157 : H7. These biochemical markers, in association with selective agents, help differentiate nonpathogenic E. coli and other commensals from most strains of E. coli O157 : H7. A second chromogenic substrate (e.g. for α or β‐galactosidase) may be used to highlight the presence of E. coli O157 amongst nonreactive background flora (Bettelheim 2005). In separate reports, Bettelheim examined the performance of three chromogenic agars; Rainbow Agar O157, CHROMagar O157 and O157 : H7 ID agar, with a large collection of verotoxigenic and nontoxigenic E. coli strains (Bettelheim 1998a,b, 2005). All three media showed high sensitivity and specificity for the recognition of E. coli O157 strains. These media are less reliable for the detection of many other verotoxigenic serotypes, e.g. E. coli O26, which typically produce β‐glucuronidase (Murinda et al. 2004). In order to detect all verotoxin producing strains, alternative strategies involving the demonstration of toxin in stool samples (e.g. by immunological techniques) or toxin genes (using nucleic amplification techniques) are preferred by some laboratories.
Detection and differentiation of yeasts
A range of chromogenic media is available for detection of yeasts. Such media have a superior ability to differentiate mixtures of different species when compared with traditional media such as Sabouraud agar (Baumgartner et al. 1996). A common principle among these media is the inclusion of a chromogenic substrate for β‐hexosaminidase thus allowing the differentiation and identification of the most frequent and clinically important species, Candida albicans. Freydière et al. (2001) have reported that detection of C. albicans via β‐hexosaminidase activity is as sensitive, more specific, less subjective and far more rapid than the conventional germ tube test. For this reason, and following a detailed cost analysis, the authors concluded that chromogenic media for isolation of yeasts are economically advantageous compared with conventional media. This finding has been supported by others (Ainscough and Kibbler 1998).
Odds and Bernaerts (1994) described the use of CHROMagar Candida, which employs a combination of chromogenic substrates to give enhanced differentiation of yeast species (see Fig. 1). As well as the differentiation of C. albicans as green colonies, this medium allows for identification of Candida tropicalis as blue colonies. Other species form either pink or white colonies. Candida krusei may be recognized by the formation of characteristic pink flat colonies although some less commonly encountered species such as Candida inconspicua have been shown to produce a similar colonial appearance (Hospenthal et al. 2006). Candida ID is another medium that contains a combination of chromogenic substrates but allows only for the identification of C. albicans. A study by Letscher‐Bru et al. (2002) with 786 clinical specimens showed that Candida ID demonstrated greater recovery of yeasts (P = 0·011) and superior identification of C. albicans (P =
