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A Decade of Development of
Chromogenic Culture Media for Clinical
Microbiology in an Era of Molecular
Diagnostics
John D. Perry
SUMMARY In the last 25 years, chromogenic culture media have found wide-
spread application in diagnostic clinical microbiology. In the last decade, the
range of media available to clinical laboratories has expanded greatly, allowing
specific detection of additional pathogens, including Pseudomonas aeruginosa, group B
streptococci, Clostridium difficile, Campylobacter spp., and Yersinia enterocolitica. New me-
dia have also been developed to screen for pathogens with acquired antimicrobial resis-
tance, including vancomycin-resistant enterococci, carbapenem-resistant Acinetobacter
spp., and Enterobacteriaceae with extended-spectrum -lactamases and carbapenemases.
This review seeks to explore the utility of chromogenic media in clinical microbiology,
with particular attention given to media that have been commercialized in the last de-
cade. The impact of laboratory automation and complementary technologies such as
matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF
MS) is also assessed. Finally, the review also seeks to demarcate the role of chromogenic
media in an era of molecular diagnostics.
INTRODUCTION
agar (21), Pourmedia Vi Candida (22), Chromogenic Candida agar (23), Brilliance Can-
dida Agar (24) and HiCrome Candida differential agar (25). A common feature of these
media is a chromogenic substrate for -hexosaminidase to discriminate C. albicans/C.
dubliniensis from other yeasts, and most include a second chromogenic substrate
(usually to detect phosphatase or -glucosidase) to provide further discrimination
between species (18). The main advantage of such chromogenic agars is their ability to
detect mixed cultures of yeasts due to the fact that different species frequently form
colonies with different colors. Such mixtures of species may be indistinguishable and
remain undetected as mixtures on conventional agars such as Sabouraud agar plus
chloramphenicol (23, 26). This is important, as different species may have different
susceptibilities to antifungal agents. While C. albicans/C. dubliniensis are usually sus-
ceptible to antifungal agents, chromogenic media may help to detect species with a
higher likelihood of resistance to azoles and/or amphotericin B, including Candida
krusei, Candida glabrata, Candida rugosa and Candida inconspicua (27).
There have been relatively few comparisons of different chromogenic agars using
clinical specimens in the last decade. Ozcan et al. compared Oxoid Chromogenic
Candida agar (OCCA) with CHROMagar Candida (CAC) and Sabouraud chloramphenicol
agar (SCA) using 392 vaginal swabs. Yeasts were isolated from 161 samples, and 21
samples (13%) yielded a mixture of species on at least one medium (23). OCCA and CAC
showed comparable sensitivity (96.9% versus 97.5%, respectively) for detection of
positive samples, whereas the sensitivity of SCA was lower (91.9%). For the 21 poly-
fungal infections, 20 (95.2%) were detected using OCCA, compared with only 14
(66.7%) using CAC (P ⬍ 0.05). Sendid et al. compared CandiSelect 4 (CS4) with CAC
using 1,549 clinical samples from a wide variety of sites (28). A total of 502 samples
(32.4%) yielded one or more yeast species, including 37 samples (7.4%) that yielded
more than one species. The sensitivities of CS4 and CAC were very similar (92.1 and
91.1%, respectively), with no false-positive results. CS4 was superior to CAC for pre-
sumptive identification of C. glabrata (80 versus 75%) and C. krusei (92 versus 83%) but
was less effective for C. tropicalis (68 versus 76%).
Pseudomonas aeruginosa
P. aeruginosa is an important nosocomial pathogen and may also cause community-
acquired infections, particularly in individuals with underlying disease. For example, in
patients with cystic fibrosis, it is a common and important cause of respiratory tract
infection. Laine et al. reported the first chromogenic medium designed specifically for
the isolation of P. aeruginosa (PS-ID), which was subsequently commercialized as
isolated as purple colonies on PS-ID, including Burkholderia cepacia complex (11). There
are no other reports of this medium with clinical samples; however, Weiser et al.
included chromID Pseudomonas in a comparison of five selective media that were
challenged with 50 isolates of P. aeruginosa and 90 isolates belonging to closely related
species (30). chromID Pseudomonas showed the highest specificity of the five media
tested, but the authors reported that its sensitivity (95%) was negatively impacted by
the large variation in color of P. aeruginosa colonies (including pink-brown and green,
possibly due to interference from natural pigments of P. aeruginosa). In conclusion,
there is a lack of any published studies with clinical samples that demonstrate a higher
recovery of P. aeruginosa using chromogenic media.
Staphylococcus aureus
S. aureus is one of the most frequent and important human pathogens and is
implicated in a range of infections, including superficial skin infections, abscesses,
bacteremia, and food poisoning. It is frequently found colonizing the nose, throat, and
TABLE 2 Summary of studies evaluating chromogenic media for the isolation of Streptococcus agalactiae from clinical samples
Positive
Sensitivity (%) Specificity (%) predictive value
Total no. of
at: at: (%) at:
Study authors, samples/no.
yr (reference) positive Swab type(s) Test medium(a)a 18–24 h 48 h 18–24 h 48 h 18–24 h 48 h
Smith et al., 200/83 Vaginal chromID Strepto B 67.5 67.5 100 100
2008 (38) CNA blood agar 57 57 89.7 89.7
Broth (CN-TH), chromID Strepto B 91.6 92.8 100 100
Broth (CN-TH), blood agar 88 89.2 88.9 89.7
Poisson et al., 528/60 Vaginal, others chromID Strepto B 71.7 90 100 85.7
2010 (47) Granada 61.7 88.3 100 100
Blood agar 46.7 66.7 82.4 88.9
Morita et al., 1,425/319 Vaginal, rectal Broth (CN-TH), chromID Strepto B 99.7
2014 (37) Broth (CN-TH), blood agar 93.7
aCNAblood agar, blood agar supplemented with colistin and nalidixic acid; broth (CN-TH), Todd-Hewitt broth supplemented with colistin and nalidixic acid; neo/nali
blood agar, blood agar supplemented with neomycin and nalidixic acid; broth (GN-TH), Todd-Hewitt broth supplemented with gentamicin and nalidixic acid.
use of a selective enrichment broth (38–44). When both types of media were tested
under the same conditions, chromogenic media showed a higher sensitivity than
selective blood agars in all of these studies. Most studies show that the sensitivity of any
culture medium for detection of GBS may be substantially improved by use of an
enrichment broth (38, 39, 41, 44, 45). Chromogenic media have a potential advantage
over Granada medium, as they do not require anaerobic incubation and have the ability
to detect nonhemolytic strains of GBS that typically fail to produce pigment on Granada
medium. Such strains are thought to account for up to 5% of invasive infections (46).
Despite this, several studies have compared Granada medium with chromogenic agars
(6, 40, 45–48), and overall there is no clear advantage of either in terms of sensitivity.
Moreover, Granada medium invariably demonstrates 100% specificity and is arguably
the only medium that can be used without the need to confirm the identification of
suspect colonies of GBS (46).
Only a few studies have compared the performance of different chromogenic media
for GBS in a head-to-head evaluation using clinical samples (46, 48, 49). No statistically
significant advantage was found for any of the media tested, and sensitivity is likely to
have been underestimated because they were not used in conjunction with an enrich-
ment broth (38, 44, 45). Most studies conclude that chromogenic media for GBS are
highly convenient tools that offer an increased sensitivity and specificity over conven-
tional blood-based media. Further large studies would be needed to establish the
superiority of any particular chromogenic medium, and the use of an enrichment broth
should ideally be included in such studies.
TABLE 3 Summary of studies comparing chromID C. difficile with other culture media for
isolation of C. difficile from stool samples.
Sensitivity
(%) at:
Study authors, yr Total no. of Sample
(reference) samples/no. positive treatment Test mediuma 24 h 48 h
Eckert et al., 2013 (70) 406/54 None chromID C. difficile 74.1 87
TCCA 85.2
CLO 70.4
Shin and Lee, 2014 (72) 530/180 Alcohol chromID C. difficile 55.6 85
CDSA 19.4 75.6
Han et al., 2014 (71) 185/36 Heat chromID C. difficile 58.3 100
CDSA 83.3
aTCCA, brain heart infusion agar plus 5% blood, taurocholate, cycloserine, and cefoxitin; CLO, Clostridium
difficile agar (bioMérieux); TCCFA, cycloserine-cefoxitin-fructose-egg yolk agar (CCFA) plus 0.1%
taurocholate; CDSA, C. difficile selective agar (BBL); CCFA, cycloserine-cefoxitin-fructose-egg yolk agar.
plus a simple disk test for pyroglutamyl aminopeptidase, and this may be useful for
laboratories without access to MALDI-TOF MS (77). Furthermore, a subset of C. difficile
strains fails to generate black colonies due to the absence of the -glucosidase gene,
and this appears to be a consistent feature of strains of ribotype 023 (78, 79). In a UK
study, the proportion of isolates failing to generate black or gray colonies within 48 h
was reported to be 1.6% (13). Such isolates may still be detected on chromID C. difficile
due to their characteristic colony shape, but care must be taken to ensure that such
colonies are not overlooked.
It is worth emphasizing that culture of C. difficile alone has little predictive value for
diagnosis of CDI without subsequent demonstration of the isolate’s ability to produce
cytotoxin. This can be directly demonstrated by testing culture supernatants on cell
lines or may be inferred much more rapidly by testing colonies for toxin genes by PCR
(80). Darkoh et al. claimed that they circumvented this problem with the report of a
new chromogenic medium (the Cdifftox plate assay) on which toxigenic C. difficile
isolates formed blue colonies, thus differentiating them from nontoxigenic isolates,
which formed white colonies (81). This was achieved using 5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside (X-Gal) for detection of the glycosyltransferase activity of toxins
A and B. Despite its promise, I am not aware of any published evaluation data for this
medium since it was first described in 2011 (81).
Campylobacter spp.
Campylobacter spp. are the most common bacterial cause of gastroenteritis (GI) in
many countries, and infection is primarily due to ingestion of contaminated food. CASA
(Fig. 1d) is a chromogenic medium initially designed for the isolation of Campylobacter
spp. from food, but it has since been evaluated with stool samples from patients with
suspected gastroenteritis. The chromogenic substrate used for detection of Campylo-
bacter spp. is undisclosed by the manufacturer. Le Bars et al. compared CASA with two
nonchromogenic agars (Karmali medium and Campylosel) for the isolation of Campy-
lobacter spp. from 370 diarrheic stool samples (14). Cultures on all three media were
incubated for up to 96 h in a microaerophilic atmosphere at two different temperatures,
37°C and 42°C. The sensitivity of CASA was equivalent to or slightly better than that
shown by either of the other two media, but CASA was reported to be much more
selective, which led to a reduction in the time required for processing colonies for
confirmation of Campylobacter. Dalziel et al. reported the culture of 979 stool samples
on CASA and modified charcoal-cefoperazone-deoxycholate agar (CCDA) with incuba-
tion of cultures at 42°C for 36 to 48 h in a microaerophilic atmosphere (82). The authors
reported sensitivities of 100% and 84% for CASA and CCDA medium, respectively (P ⬍
0.001), and also reported a higher selectivity of CASA. A limitation of CASA is the lack
of a specific chromogenic reaction to indicate the presence of Campylobacter spp., as
other species that grow on the medium also hydrolyze the chromogenic substrate to
produce pink or red colonies, and some may therefore appear quite similar to Campy-
lobacter spp.
Salmonella spp.
Salmonella spp. remain one of the most important causes of foodborne gastroenteritis,
and chromogenic media designed for the specific isolation of Salmonella spp. have been
available for at least 25 years. Rambach agar and SM-ID were the first two examples of such
media (1), and both have been superseded by new generations of media. The principles of
Shigella spp.
Shigella spp. produce few hydrolytic enzymes that provide useful differentiation
from other bacteria, and that has restricted the development of chromogenic media for
their detection. However, the observation that all species of Shigella produce
Vibrio spp.
Media for the isolation of pathogenic Vibrio spp., which have been designed
primarily for screening food samples, have been evaluated for use with clinical samples
(15, 92). CHROMagar Vibrio and chromID Vibrio both allow for the isolation and
differentiation of the two most important pathogenic species, Vibrio cholerae and Vibrio
parahaemolyticus, and accommodate isolation of other Vibrio species. The chromogenic
substrates used in these media are undisclosed by the manufacturers. CHROMagar
Yersinia enterocolitica
Y. enterocolitica is a foodborne pathogen and a cause of diarrhea and pseudoap-
pendicitis. The most widely used conventional agar for detection of this pathogen in
stool samples is cefsulodin-irgasan-novobiocin (CIN) agar, which utilizes mannitol
fermentation as a biochemical indicator for Y. enterocolitica. CIN agar is effective, but its
specificity is limited as a number of other species of Enterobacteriaceae are able to grow
on the medium and ferment mannitol, thus also generating magenta colonies (e.g.,
Serratia spp., Providencia spp., Klebsiella oxytoca, and Citrobacter freundii). Weagant
described the development of Yersinia enterocolitica chromogenic medium (YeCM)
(93). This medium sought to improve on the specificity of CIN agar by utilizing
cellobiose as the fermentable carbohydrate for detection of Y. enterocolitica and by
including a chromogenic substrate for -glucosidase, an enzyme that is produced by
most other species of Enterobacteriaceae that are able to grow on CIN agar (93). As well
as enabling differentiation of Y. enterocolitica from other species, this medium had the
additional advantage that only pathogenic types of Y. enterocolitica would be detected
(as nonpathogenic biovars produce -glucosidase). There are no reports of the use of
this medium with human clinical samples, although YeCM was used in a later study that
examined the presence of Y. enterocolitica in 900 tonsil swabs from pigs (94).
Renaud et al. described the use of CHROMagar Yersinia (CAY) (Fig. 1e) for the
isolation of Y. enterocolitica from 1,494 stool samples from hospitalized patients and
used CIN agar as a comparator (17). Although the composition of this medium is
undisclosed, the medium achieves outcomes very similar to those obtained with YeCM,
suggesting the inclusion of a substrate for -glucosidase to increase specificity (95). Six
isolates of pathogenic Y. enterocolitica were successfully isolated using both CAY and
CIN agar, but CAY showed a much higher specificity (99%) than CIN agar (90.4%), with
only 14 false-positive isolates recovered on CAY (P ⬍ 0.001). In contrast, 137 isolates
belonging to other species (predominantly C. freundii and Providencia spp.) grew as
false-positive colonies on CIN agar, and there was no differentiation between the six
pathogenic Y. enterocolitica isolates recovered and six additional nonpathogenic iso-
lates of biovar 1A on CIN agar. Another chromogenic agar, Yersinia Enterocolitica Agar
(YECA), has been described, but its composition is undisclosed and there are no data
available for testing of human samples (96).
TABLE 4 Summary of selected studies evaluating chromogenic media for the isolation of MRSA from patient samples
Sensitivity (%) Specificity (%)
at: at:
Study authors, yr Total no. of
(reference) samples/no. positive Sample type(s) Test medium 24 h 48 h 24 h 48 h
Yang et al., 578/99 Nasal swabs MSA-Fxa 92.9 96 97.1 95.2
2010 (106) MRSASelect 94.9 100 98.5 97.7
MRSA-ID 90.9 99 98.1 97.9
CHROMagar MRSA 91.9 99 99.5 99
Morris et al., 6,035/147 Nasal, groin, axilla, and Brilliance MRSA 2 78.2 99.9
2012 (107) wound swabs chromID MRSA 93.2 99.8
chromID MRSA 2 83.7 99.9
Colorex MRSA 87.1 99.9
Denys et al., 515/73 Nasal swabs BBL CHROMagar MRSA II 87.7 98.6
2013 (108) MRSASelect 89 93.4
Spectra MRSA 83.6 92.1
that the use of broth enrichment prior to inoculation onto chromogenic agar can
significantly increase the sensitivity of MRSA detection, and this is exemplified by two
of the studies in Table 4 (109, 110).
Vancomycin-Resistant Enterococci
chromID VRE was the first chromogenic medium reported for the isolation of
enterococci with acquired resistance to glycopeptides (e.g., vancomycin and teicopla-
nin) (8). In six studies with stool samples or rectal swabs, chromID VRE was compared
with bile-esculin agars (with or without azide) supplemented with vancomycin (8,
111–115). Two of these studies reported a superior sensitivity of chromID VRE (8, 115),
and the others reported equivalent sensitivity (111–114). A consistent advantage of
chromID VRE was its ability to differentiate between Enterococcus faecalis and Entero-
coccus faecium. This is achieved by incorporating chromogenic substrates for detection
of ␣-glucosidase and -galactosidase (18). chromID VRE also showed a higher speci-
ficity, leading to a reduction in the number of confirmatory tests that were required for
suspect colonies. Five other chromogenic media, AES VRE agar (115), Brilliance VRE (Fig.
2a) (116), CHROMagar VRE (117, 118), Spectra VRE (119–121), and VRESelect (122), have
since been evaluated against bile-esculin agars (with or without azide) supplemented
with vancomycin, in studies with stool samples or rectal swabs. In each of the eight
cited studies, the chromogenic media offered higher sensitivity and specificity. All
except AES VRE agar provided differentiation of E. faecalis from E. faecium.
Relatively few studies have included head-to-head comparisons of chromogenic
agars for the isolation of VRE from clinical samples (115, 123–125). CHROMagar VRE and
chromID VRE were evaluated with 259 stool samples after an overnight enrichment
step and a 48-h incubation period (123). The authors reported an identical sensitivity of
the two media (98.2%) and an equivalent specificity. Suwantarat et al. performed a
TABLE 5 Summary of studies evaluating chromogenic media for the isolation of Enterobacteriaceae with extended-spectrum--lactamases
from clinical samples
Positive
Sensitivity (%) predictive value
at: (%) at:
Study authors, yr Total no. of
(reference) samples/no. positive Sample type(s) (n) Test medium 18–24 h 48 h 18–24 h 48 h
Réglier-Poupet et al., 765/33 Rectal swab (468), urine chromID ESBL 88 94 39 28
2008 (128) (255), respiratory (42) BLSE 85 85 15 11
Huang et al., 528/59 Fecal (344), respiratory (134), chromID ESBL 94.9 48.7
2010 (129) miscellaneous (50) Brilliance ESBL 94.9 46.7
MAC with ceftazidime disk 74.6 64.7
Willems et al., 139/16 Perineal and nasal swabs chromID ESBL 81.2 87.5 35.1 33.3
2013 (132) (139) Brilliance ESBL 87.5 87.5 38.9 33.3
BLSE 87.5 87.5 22.2 20.3
Grohs et al., 2,337/354 Rectal swab (2,337) chromID ESBL 97.5 39.1
2013 (133) Brilliance ESBL 98.6 29.5
CHROMagar ESBL 98.3 38.7
Drigalski ⫹ CAZ 97.2 32.5
Drigalski ⫹ CTX 95.5 29.5
Extended-Spectrum--Lactamase-Producing Enterobacteriaceae
Extended-spectrum -lactamases (ESBLs) have become globally disseminated within
species of Enterobacteriaceae. These enzymes hydrolyze third-generation cephalosporins,
typically conferring resistance to these agents, but are inhibited by clavulanic acid. Genes
encoding ESBLs are frequently found on transmissible plasmids that often harbor other
resistance determinants (e.g., encoding resistance to aminoglycosides). Prompt and accu-
rate detection of ESBL producers can assist in guiding optimal antimicrobial therapy (126)
and limiting nosocomial transmission (127). The first published report of a chromogenic
medium for detection of ESBL producers described the evaluation of ESBL-Bx (a prototype
of chromID ESBL). The authors compared this new medium with MacConkey agar supple-
mented with 2 g/ml ceftazidime for the isolation of ESBL producers from 644 clinical
samples (7). They reported a sensitivity of 97.7% for the chromogenic medium, compared
with 84.1% for MacConkey agar plus ceftazidime. Since this first report, a range of chro-
mogenic media has been made commercially available, and reports of their performance
are summarized in Table 5 (128–134). The principles of these media have much in
common. The media typically employ a combination of chromogenic substrates to
detect -galactosidase (or -glucuronidase) production by E. coli and -glucosidase
production by the KES group. This allows detection and differentiation of the main
species or groups associated with ESBL production. A cephalosporin is incorporated
to inhibit susceptible strains, and other inhibitors may be included to inhibit the
growth of Gram-positive bacteria and yeasts. Enterobacteriaceae with AmpC
-lactamase are frequently isolated on all of these media, and to a large degree this
accounts for the relatively low positive predictive values shown in Table 5 (133).
Five of the studies in Table 5 include a direct comparison of two or more chromo-
genic media, and no significant difference in sensitivity was reported after 24 h of
incubation in any of these studies (129, 131–134). The yield of ESBL producers may be
increased significantly (particularly on chromID ESBL) by extending incubation up to 48
h (128, 134), and a preenrichment step may also significantly increase sensitivity (134).
However, both of these strategies contribute to an even lower specificity (132, 134). In
three of the studies in Table 5 the positive predictive value of chromogenic media was
reported to be significantly higher than that of nonchromogenic comparators (128, 130,
132).
Carbapenemase-Producing Enterobacteriaceae
Perhaps the most significant development in clinical bacteriology over the last
decade has been the global proliferation of Enterobacteriaceae with acquired carbap-
enemase enzymes (carbapenemase-producing Enterobacteriaceae [CPE]) (135). Avail-
able data suggest that the vast majority of carbapenemases in Enterobacteriaceae
belong to one of the five major families: the IMP, NDM, and VIM metalloenzymes and
the Klebsiella pneumoniae carbapenemase (KPC) and OXA-48-like enzymes (136). CPE
TABLE 6 Summary of studies evaluating chromogenic media for the isolation of carbapenemase-producing Enterobacteriaceae from
patient samples
Study authors, yr Total no. of Sensitivity Specificity Study Dominant
(reference) samples/no. positive Test medium (%) (%) location enzyme(s)
Samra et al., 2008 (9)a 122/41 CHROMagar KPC 100 98.4 Israel KPC
MacConkey agar ⫹ carbapenem disks 92.7 95.9
Adler et al., 2011 (152) 139/33 CHROMagar KPC 84.9 88.7 Israel KPC
MacConkey agar ⫹ carbapenem disks 75.8 89.6
MacConkey agar ⫹ imipenem (1 g/ml) 84.9 94.3
Panagea et al., 2011 126/46 CHROMagar KPC 97.8 Greece KPC, VIM
(153) MacConkey agar ⫹ imipenem (1 g/ml) 78.3
Vrioni et al., 2012 (154) 200/73 TSBb ⫹ ertapenem (2 g/ml) 89.1 86.4 Greece KPC, VIM
chromID ESBL 92.4 93.3
chromID ESBL (plus enrichment) 92.4 84.7
Vasoo et al., 2014 (155) 150/47 CHROMagar KPC 76.6 75.7 USA KPC
Remel Spectra CRE 97.8 86.4
MacConkey agar ⫹ ertapenem disks 83 73.8
Zarakolu et al., 2015 302/33 chromID CARBA 57.6 98.9 Turkey OXA-48
(157) chromID OXA-48 75.8 99.3
TSB ⫹ ertapenem (2 g/ml) 57.6 95.2
samples. Consequently, the values for sensitivity and specificity reported in evaluations
with clinical samples are usually lower than those achieved with pure cultures (140).
Table 6 summarizes the findings of the largest studies that have been performed with
fecal samples (stool samples or rectal swabs) from patients (9, 152–159). In three early
studies with CHROMagar KPC (9, 152, 153), sensitivity was equivalent to or higher than
that of in-house preparations of MacConkey agar incorporating imipenem (or Mac-
Conkey agar with carbapenem disks). Later studies revealed that isolates producing
NDM-1 carbapenemase may be inhibited by CHROMagar/Colorex KPC if the isolates
have a relatively low MIC to meropenem (ⱕ2 g/ml) (141, 143). This emphasizes the
importance of performing studies in different geographical areas where different
carbapenemase enzymes may predominate. In a further example, chromID CARBA was
proven to be effective in comparative studies of media in Greece (154, 156) and
Pakistan (141, 160, 161) but had limited efficacy in Turkey (157) and Belgium (162),
where OXA-48 is the dominant carbapenemase. To address this, the manufacturer
offers a biplate (chromID CARBA SMART) that combines two media in a single petri dish:
chromID CARBA and chromID OXA-48.
Brilliance CRE agar is marketed for the isolation of carbapenem-resistant Enterobac-
teriaceae (CRE) rather than CPE. This medium was evaluated in two simultaneous
studies in two centers in Pakistan and showed a lower sensitivity and specificity than
chromID CARBA for detection of CPE with NDM-1 carbapenemase (160, 161). The
authors speculated that the stability of Brilliance CRE may have been compromised
during transport of the culture media from the UK to Pakistan. However, in challenge
experiments with pure cultures, a relatively low specificity of 71% was recorded for this
medium due to the growth of AmpC- and/or ESBL-producing isolates (146). In another
study with Brilliance CRE with patient samples (n ⫽ 77), the specificity of Brilliance CRE
was lower than that of SuperCarba (86.6% versus 98.5%, respectively) (149). However,
the sensitivity of Brilliance CRE was significantly better than that of chromID CARBA for
detection of OXA-48 as shown by a study in Belgium (162). The sensitivity of both
media was improved by preenrichment of rectal swabs in MacConkey broth (although
this was not statistically significant), whereas prolonged incubation of media for 48 h
showed no advantage.
SuperCarba medium is a nonchromogenic medium for isolation of CPE that incor-
porates a low concentration of ertapenem (0.5 g/ml) in addition to cloxacillin in a
bacter spp. or it can be further supplemented to detect only isolates with resistance to
carbapenems (10). The chromogenic substrate used for detection of Acinetobacter spp.
is undisclosed by the manufacturer. The authors evaluated the supplemented version
of CHROMagar Acinetobacter for detection of carbapenem-resistant strains from stool
samples and perineal swabs of 70 patients and compared its performance to PCR
following enrichment culture. The sensitivity and specificity of the chromogenic me-
dium compared with the PCR assay were reported as 91.7% and 89.6%, respectively
(10).
There have been few subsequent published evaluations of CHROMagar Acinetobac-
ter with clinical samples for the isolation of carbapenem-resistant isolates. Song et al.
used an updated version of the medium (Fig. 1f) for isolation of carbapenem-resistant
Acinetobacter from 406 paired nasal and rectal swabs (168). They confirmed the high
specificity of the medium for carbapenem-resistant Acinetobacter, but no comparator was
used for assessment of sensitivity. In a study using spiked stool samples, CHROMagar
culture media, and chromogenic media in particular, in the clinical laboratory. These
include automated methods for inoculation of culture plates (177) and methods for
automated digital analysis that enable detection and enumeration of colored colonies
(178, 179). Digital imaging software is capable of distinguishing differences in pixel
color, and chromogenic media are particularly suited to digital automation, as color
thresholds can be created to detect target growth of specific pathogens (179). Faron et
al. exploited this technology to screen for MRSA in a multicenter study using the
WASPLab chromogenic detection module (179). Three different chromogenic media,
MRSASelect, CHROMagar MRSA, and chromID MRSA, were used, and cultures from
57,690 screening swabs were interpreted by manual reading of digital images and by
automated digital analysis. Automated analysis demonstrated 100% sensitivity for all
three chromogenic media and 90.7% specificity, indicating that automated analysis was
highly reliable for reporting negative samples (which constituted 88.6% of total sam-
ples in this study). Technologist support was required for examination of cultures
to any form of culture, as discussed above. De Boer et al. employed a multiplex PCR
assay to target four bacterial gastrointestinal pathogens (Salmonella spp. Campylobac-
ter jejuni, STEC, and Shigella/enteroinvasive E. coli) in 13,974 stool samples. They
reported that the molecular assay markedly improved rates of detection of all patho-
gens apart from Salmonella spp. compared to culture on conventional media (191). The
use of commercial platforms that employ multiplexed molecular assays and can
accommodate a large number of targets is an increasingly attractive option to simplify
laboratory workflow. One example of such a platform is the FilmArray GI panel, which
consists of automated nucleic acid extraction, reverse transcription, amplification, and
analysis and can detect 22 pathogens, with results available within 1 h. In a multicenter
study, Buss et al. examined the performance of this panel for detection of a range of GI
pathogens in 1,556 stool samples (192). Using conventional culture (with nonchromo-
genic media) as a comparator, the molecular assay showed high sensitivity (94.5 to
100%) for detection of Salmonella, Yersinia, STEC, Campylobacter, C. difficile, and Shi-
as PCR. In a study in Belgium, Huang et al. applied the Check-Direct CPE PCR assay for
the testing of 394 rectal swabs to detect CPE carriage and also cultured the swabs on
two chromogenic media (chromID CARBA/chromID OXA-48) (205). They reported that
CPE was detected by PCR in 38 samples but that only 17 samples yielded CPE by
culture. OXA-48 was the dominant carbapenemase enzyme. Of the 21 samples that
were positive by PCR but negative on culture, five of the samples were from patients
with previous carriage of CPE, and a further six samples (all with OXA-48 detected by
PCR) were from patients located in a center where a longstanding outbreak of OXA-
48-producing Klebsiella pneumoniae was occurring. It is possible that this study dem-
onstrates a superior sensitivity of PCR for detection of CPE, although the possibility that
carbapenemase genes were detected in species other than Enterobacteriaceae that may
not be targeted by chromogenic media cannot be excluded (206, 207). Otter et al.
performed screening of 4,006 patients for CPE using PCR (Check-Direct CPE assay) and
culture on a chromogenic medium (chromID CARBA SMART) at a hospital in London, UK
Salmonella” (194). In the long term, metagenomic approaches may allow reference
laboratories to type enteropathogens directly from clinical samples, but until such tests
are widely available, it is essential that culture methods are utilized for recovery of
pathogens to allow for typing of isolates and investigation of outbreaks. For such
culture methods, the relative attributes of chromogenic versus conventional agars
should be carefully considered.
CONCLUSIONS
The last decade has seen a rapid expansion in the range of chromogenic culture
media available to clinical laboratories. In most cases, a clear advantage over conven-
tional culture media can be demonstrated. This not only is due to the inclusion of
chromogenic substrates that allow detection of pathogens with high specificity but
also is due to the inclusion of optimal combinations of selective agents that prevent
interference from nontarget microorganisms. In some cases, media that have been
ACKNOWLEDGMENTS
The Freeman Hospital Microbiology Department has received funding from bio-
Mérieux for the development and evaluation of culture media, and I have performed
consultancy work for the same company. The Freeman Hospital Microbiology Depart-
ment has also received funding for evaluation studies from Bio-Rad Laboratories and
International Diagnostics Group.
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